KR20230119130A - Methods of Treating Metabolic Disorders and Cardiovascular Disease with Inhibin Subunit Beta E (INHBE) Inhibitors - Google Patents

Abstract

The present disclosure provides methods for treating a subject having a metabolic disorder and/or cardiovascular disease, methods for identifying a subject at increased risk of developing a metabolic disorder and/or cardiovascular disease, and human inhibin subunit beta E variant nucleic acid molecules. and methods for detecting variant polypeptides.

Description

Methods of Treating Metabolic Disorders and Cardiovascular Disease with Inhibin Subunit Beta E (INHBE) Inhibitors

Reference to Sequence Listing

This application contains a sequence listing submitted electronically as a text file named 18923805702SEQ, 28 kilobytes in size, created on December 11, 2021. This sequence listing is incorporated herein by reference.

The present disclosure generally relates to methods of treating a subject having a metabolic disorder and/or cardiovascular disease with an inhibin subunit beta E inhibitor, methods of identifying a subject at increased risk of developing a metabolic disorder and/or cardiovascular disease, and INHBE Variant nucleic acid molecules and methods of detecting variant polypeptides.

Body fat distribution is an important risk factor for cardiovascular and metabolic diseases independent of overall adiposity. characterized by greater fat accumulation around the waist (eg, greater abdominal fat or greater waist circumference) and/or lower peri-hip fat accumulation (eg, lower thigh fat or smaller hip circumference) Body fat distribution results in a greater waist-to-hip ratio (WHR) and is associated with a higher cardio-metabolic risk independently of body mass index (BMI). Metabolic conditions associated with body fat distribution include type 2 diabetes mellitus, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, and very-low-density lipoprotein cholesterol (VLDL-C)), apolipoprotein B or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (e.g., inability to accumulate fat in local fat depots (partial lipodystrophy) or throughout the body (lipotrophy)), insulin resistance, or during fasting or High or altered insulin levels during metabolic attempts, liver fat deposition or fatty liver disease and its complications (eg, cirrhosis, fibrosis or inflammation of the liver), non-alcoholic steatohepatitis, other types of liver inflammation, higher, elevated or altered liver Enzyme levels or other markers of liver damage, inflammation or fat deposition in the liver, high blood pressure and/or hypertension, hyperglycemia or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the aforementioned conditions This includes, but is not limited to. Identifying genetic variants associated with more favorable fat distribution (e.g., lower WHR, especially when adjusted for BMI) is a pathway to identifying mechanisms that may be exploited therapeutically for these cardiovascular-metabolic disease benefits. It can be.

Inhibin subunit beta E (INHBE) is a member of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Inhibin has been implicated in the regulation of numerous cellular processes including cell proliferation, apoptosis, immune response and hormone secretion. Inhibin and activin inhibit and activate the secretion of foliotropin by the pituitary gland, respectively. Depending on their subunit composition, inhibins/activins regulate various functions such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, neuronal cell survival, embryonic axis development or bone growth. get involved in Inhibin appears to oppose the action of activin. In addition, INHBE can be upregulated under conditions of endoplasmic reticulum stress, and this protein can inhibit cell proliferation and growth in the pancreas and liver.

The present disclosure provides methods of treating a subject having, or at risk of developing, a metabolic disorder, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having type 2 diabetes or at risk of developing type 2 diabetes comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having obesity or at risk of developing obesity comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having elevated triglyceride levels (hypertriglyceridemia) or at risk of developing elevated triglyceride levels (hypertriglyceridemia) comprising administering to the subject an INHBE inhibitor. do.

The present disclosure also provides methods of treating a subject having lipodystrophy or at risk of developing lipodystrophy, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing liver inflammation comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing fatty liver disease comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having hypercholesterolemia or at risk of developing hypercholesterolemia comprising administering to the subject an INHBE inhibitor.

The present disclosure also relates to having or having elevated liver enzymes (eg, alanine transaminase (ALT) and/or aspartate transaminase (AST)), comprising administering an INHBE inhibitor to a subject. Methods of treating a subject at risk for developing liver enzymes (eg, ALT and/or AST) are provided.

The present disclosure also provides a method of treating a subject having non-alcoholic steatohepatitis (NASH) or at risk of developing NASH, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject suffering from or at risk of developing a cardiovascular disease comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having cardiomyopathy or at risk of developing cardiomyopathy, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing heart failure comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides a method of treating a subject having or at risk of developing hypertension, comprising administering an INHBE inhibitor to the subject, comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits a metabolic disorder, wherein the subject is suffering from a metabolic disorder, the method comprising obtaining or obtaining a biological sample from the subject; Determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by performing or performing a genotyping assay on the biological sample to determine whether the subject has a genotype comprising the INHBE variant nucleic acid molecule. includes; If the subject is on INHBE criteria, administering or continuing administration of a therapeutic agent that treats or inhibits the metabolic disorder at a standard dose to the subject, administering an INHBE inhibitor to the subject; If the subject is heterozygous for the INHBE variant nucleic acid molecule, administering or continuing to administer to the subject a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or lower than the standard dose, administering to the subject an INHBE inhibitor; If the subject is homozygous for the INHBE variant nucleic acid molecule, administering or continuing administration of a therapeutic agent that treats or inhibits the metabolic disorder to the subject in an amount equal to or lower than the standard dose; Herein, the presence of a genotype having an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing a metabolic disorder.

The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits a cardiovascular disease, wherein the subject is suffering from a cardiovascular disease, the method comprising obtaining or obtaining a biological sample from the subject; Determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by performing or performing a genotyping assay on the biological sample to determine whether the subject has a genotype comprising the INHBE variant nucleic acid molecule. includes; If the subject is on INHBE criteria, administering or continuing administration of a therapeutic agent for treating or inhibiting cardiovascular disease at a standard dose to the subject, administering an INHBE inhibitor to the subject; If the subject is heterozygous for the INHBE variant nucleic acid molecule, administering or continuing to administer to the subject a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or lower than the standard dose, administering to the subject an INHBE inhibitor; If the subject is homozygous for the INHBE variant nucleic acid molecule, administering or continuing to administer to the subject a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or lower than the standard dose; Here, the presence of a genotype having an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing cardiovascular disease.

The present disclosure also provides a method for identifying a subject at increased risk of developing a metabolic disorder, wherein the method determines the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from the subject. includes steps that have been or have been determined; wherein if the subject is on INHBE criteria, the subject has an increased risk of developing a metabolic disorder; When a subject is heterozygous for the INHBE variant nucleic acid molecule or homozygous for the INHBE variant nucleic acid molecule, the subject has a reduced risk of developing a metabolic disorder.

The present disclosure also provides a method for identifying a subject at increased risk of developing cardiovascular disease, wherein the method determines the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from the subject. includes steps that have been or have been determined; wherein if the subject is on INHBE criteria, the subject has an increased risk of developing cardiovascular disease; When a subject is heterozygous for the INHBE variant nucleic acid molecule or homozygous for the INHBE variant nucleic acid molecule, the subject has a reduced risk of developing cardiovascular disease.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides; INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or a therapeutic agent that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides; INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or a therapeutic agent for treating or inhibiting cardiovascular disease for use in the treatment of cardiovascular disease in a subject having an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides; INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or an INHBE inhibitor that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides; INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or an INHBE inhibitor for treating or inhibiting cardiovascular disease for use in the treatment of cardiovascular disease in a subject having an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the detailed description serve to explain the principles of the present disclosure.
Figure 1 shows the association of INHBE predicted loss-of-function (pLOF) variants with favorable fat distribution (i.e., low BMI-adjusted WHR) in exome sequencing analysis of over 525,000 people in multiple studies; Association analysis was estimated by fitting a mixed-effects linear regression model accounting for association and population stratification using REGENIE software; Abbreviations: confidence interval, CI; standard deviation, SD; body mass index, BMI; waist-to-hip ratio adjusted for BMI, WHRadjBMI; reference-reference allele, RR; reference-replacement allele, RA; replacement-replacement allele, AA; UK Biobank Cohort, UKB; European ancestry, EUR; Mexico City Prospective Study Cohort, MCPS; Predicted loss-of-function, pLOF.
Figure 2 depicts a genetic model of INHBE showing the location of pLOF variants (top panel) and the phenotypic distribution of BMI-adjusted WHR in carriers of each variant; Blue bars show median BMI-adjusted WHR for non-bearers, red bars show median BMI-adjusted WHR for bearers; The two variants highlighted in dark red were individually associated with low BMI-adjusted WHR; Data are from the UK Biobank (UKB) and Mexico City Prospective Study (MCPS) cohorts. Abbreviations: Body Mass Index, BMI; Waist-hip ratio, WHR.
Figure 3 shows the in silico predicted functional consequences of the INHBE c.299-1G:C (12:57456093:G:C) splice variant; top sequence = original exon 2 (SEQ ID NO: 28); Subsequence = predicted exon 2 (SEQ ID NO: 29).
Figure 4 shows the wild-type INHBE protein sequence (top; SEQ ID NO: 8) and the in silico predicted protein sequence for the c.299-1G:C receptor splice variant (bottom; SEQ ID NO: 8, changes in unhighlighted regions). shows).
Figure 5 shows Chinese Hamster Ovary (CHO) cell experiments for the c.299-1G>C variant. The variant occurs at the splice acceptor site for the first and only splice junction of the INHBE gene (Panel A). In CHO cells, the c.299-1G>C variant results in the expression of a low molecular weight variant present in cell lysates but not media, consistent with loss-of-function (Panel B).
Figure 6 shows the association of INHBE pLOF variants with body fat and lean mass, percentage and body surface-adjusted index measured by electrical bioimpedance in 423,418 UKB study participants.
Figure 7 shows INHBE expression patterns across tissues (left, Figure 7a) and liver cell types (right, Figure 7b). The first panel shows normalized mRNA expression values for INHBE by tissue using data from the Genotype Tissue Expression (GTEx) Consortium (GTEx Portal 2021. Accessed 2021, June 1 ^st via world wide web at "gtexportal.org/"). Shown in parts per million (CPM). The second panel is normalized cell-type specific in liver in transcripts per million protein-coding genes (pTPM) obtained from the Human Protein Atlas (HPA) (Uhlen et al., Nat. Biotechnol. 2010, 28, 1248-50). show the expression level. Box plots represent median (thick black vertical bars), interquartile range, minimum and maximum CPM values across subjects per tissue.
8 shows that hepatic mRNA expression of INHBE is upregulated in patients with steatosis and non-alcoholic steatohepatitis (NASH) compared to subjects with normal livers in bariatric surgery patients with GHS. In the top panel ( FIG. 8A ), the plots show normal liver (control), hepatic steatosis (simple steatosis) and non-alcoholic steatohepatitis (NASH) patients per million transcripts (TPM; RNA for one million molecules detected in a particular experiment). Normalization of molecules) shows the hepatic mRNA expression level of INHBE. The bottom panel ( FIG. 8B ) has statistics for comparison between groups. The simple steatosis group showed higher INHBE expression in the liver than the control group. The NASH group showed higher expression compared to both the control group and the simple steatosis group. All differences in expression between groups were statistically significant.

Various terms relating to aspects of the present disclosure are used throughout the specification and claims. These terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms shall be construed in a manner consistent with the definitions provided herein.

Unless otherwise specified, in no way should any method or aspect described herein be construed as requiring that the steps be performed in any particular order. Thus, if a method claim does not specifically state in a claim or description that steps are limited to a particular order, order is not intended to be inferred in any way. This applies to all possible unexpressed grounds for interpretation, including the number or type of aspects described in the specification, or the general meaning derived from logical problems, grammatical constructions or punctuation regarding the arrangement of steps or flow of operations.

As used herein, the singular forms “a” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term “about” means that the recited numbers are approximate and that small changes do not materially affect the practice of the disclosed embodiments. When a number is used, unless indicated otherwise by context, the term "about" means that the number can vary by ±10% and remain within the range of the disclosed embodiments.

As used herein, the term "comprising" may be replaced with "consisting of" or "consisting essentially of" in certain embodiments, if desired.

The term “isolated” as used herein with reference to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is in a condition other than its natural environment, such as away from blood and/or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, a nucleic acid molecule or polypeptide may be in highly purified form, i.e., at least 95% pure or at least 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" or "oligonucleotide" may include polymeric nucleotides of any length, including DNA and/or RNA. It can be single-stranded, double-stranded or multi-stranded. One strand of a nucleic acid also refers to its complement.

As used herein, the term “subject” includes all animals including mammals. Mammals include farm animals (eg horses, cows, pigs), companion animals (eg dogs, cats), laboratory animals (eg mice, rats, rabbits) and non-human primates, but , but not limited thereto. In some embodiments, the subject is a human. In some embodiments, the human is a patient in the care of a physician.

In accordance with the present invention, loss-of-function variants of INHBE (whether such variants are homozygous or heterozygous in a particular subject) are associated with metabolic disorders such as type 2 diabetes, obesity, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, observed to be associated with elevated liver enzymes (e.g., ALT and/or AST), NASH and/or elevated triglyceride levels, and/or reduced risk of developing cardiovascular disease, e.g., cardiomyopathy, heart failure, hypertension It became. Loss-of-function variants of the INHBE gene or protein are not believed to be associated with metabolic disorders and/or cardiovascular disease in genome-wide or exome-wide association studies. Thus, a subject who is homozygous or heterozygous for a reference INHBE variant nucleic acid molecule can be treated with an INHBE inhibitor such that the metabolic disorder and/or cardiovascular disease is inhibited, symptoms thereof are reduced, and/or development of symptoms is inhibited. In addition, subjects with metabolic disorders and/or cardiovascular disease may have type 2 diabetes, obesity, hypertension, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (e.g., ALT and/or AST), NASH and/or metabolic disorders such as elevated triglyceride levels, and/or cardiovascular diseases such as cardiomyopathy, heart failure and hypertension.

For purposes of this invention, any particular subject, such as a human, can be classified as having one of three INHBE genotypes: i) INHBE criteria; ii) heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; or iii) homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. If the subject does not have a copy of the INHBE variant nucleic acid molecule encoding the INHBE predicted loss-of-function polypeptide, then the subject is INHBE criteria. A subject is heterozygous for the INHBE variant nucleic acid molecule if the subject has a single copy of the INHBE variant nucleic acid molecule encoding the INHBE predicted loss-of-function polypeptide. An INHBE variant nucleic acid molecule is any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule) encoding an INHBE polypeptide having partial loss-of-function, complete loss-of-function, predicted partial-loss-of-function, or predicted complete loss-of-function. or a cDNA molecule). A subject having an INHBE polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for INHBE. When a subject has two copies (identical or different) of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is homozygous for the INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In the case of a subject genotyped or determined on INHBE criteria, such subject has a metabolic disease, e.g., type 2 diabetes, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (e.g., ALT and and/or AST), obesity, high blood pressure and/or elevated triglyceride levels (hyperglyceridemia) and/or an increased risk of developing cardiovascular diseases such as cardiomyopathy, heart failure and high blood pressure. For subjects genotyped or determined to be INHBE criteria or heterozygous for an INHBE variant nucleic acid molecule, such subject or subjects may be treated with an INHBE inhibitor.

In any of the embodiments described herein, an INHBE variant nucleic acid molecule is any nucleic acid molecule that encodes an INHBE polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function (e.g., eg, genomic nucleic acid molecules, mRNA molecules or cDNA molecules). In some embodiments, the INHBE variant nucleic acid molecule is associated with a reduced in vitro response to an INHBE ligand compared to a reference INHBE. In some embodiments, an INHBE variant nucleic acid molecule is an INHBE variant that results in or is predicted to result in premature cleavage of an INHBE polypeptide compared to a human reference genomic sequence. In some embodiments, an INHBE variant nucleic acid molecule is a variant predicted to be impaired by an in vitro prediction algorithm such as Polyphen, SIFT or similar algorithm. In some embodiments, an INHBE variant nucleic acid molecule is a variant that causes or is predicted to cause a nonsynonymous amino acid substitution in INHBE and whose allele frequency is less than 1/100 allele in a population from which the subject is selected. In some embodiments, an INHBE variant nucleic acid molecule is any rare missense variant (allele frequency < 0.1%; or 1 in 1,000 alleles), or any splice-site, stop-gain, start-loss, stop-loss, frameshift, or inframe indel or other frameshift INHBE variants.

In any of the embodiments described herein, the INHBE predicted loss-of-function polypeptide can be any INHBE polypeptide having partial loss-of-function, complete loss-of-function, predicted partial-loss-of-function, or predicted complete loss-of-function. .

In any of the embodiments described herein, the INHBE variant nucleic acid molecule encoding a variant in protein sequence is an INHBE reference genomic nucleic acid molecule (SEQ ID NO: 1; ENST00000266646.3 chr12:57455307- in GRCh38/hg38 human genome assembly) as a reference sequence. 57458025) can be used to include mutations at locations on chromosome 12.

There are numerous genetic variations of INHBE that cause subsequent changes in the INHBE polypeptide sequence, including but not limited to: Gln7fs, Arg18STOP, Gln37STOP, Arg40STOP, Leu55fs, Cys139fs, Arg144STOP, Cys192fs, Arg224fs, Arg224STOP, Arg233fs, Arg250STOP, Asp251fs, Tyr253STOP, Tyr275STOP, Ser293fs, Trp308fs, Pro309fs, Arg320STOP, Leu323fs, and Ter351Tyrext*?. Additional variant genomic nucleic acid molecules of INHBE (using human genome reference construction GRch38) include C298+1G:T (12:57455835:G:T), c.299-2A:G, c.299-1G:C (12 :57456093:G:C), and 12:57259799:A:C. Additional variant INHBE polypeptides exist, including but not limited to INHBE polypeptides in which the methionine at position 1 is removed.

Any one or more (i.e., any combination) of INHBE pLOF variants can be used within any of the methods described herein to determine whether a subject has an increased risk of developing a metabolic disorder and/or cardiovascular disease. . Combinations of specific variants may form a mask used for statistical analysis of specific correlations between INHBE and increased type 2 diabetes/BMI risk and/or cardiovascular disease.

In any of the embodiments described herein, the metabolic disorder is type 2 diabetes, obesity, NASH, and/or elevated triglyceride levels. In any of the embodiments described herein, the metabolic disorder is type 2 diabetes. In any of the embodiments described herein, the metabolic disorder is obesity. In any of the embodiments described herein, the metabolic disorder is NASH. In any of the embodiments described herein, the metabolic disorder is elevated triglyceride levels. In any of the embodiments described herein, the metabolic disorder is lipodystrophy. In any of the embodiments described herein, the metabolic disorder is liver inflammation. In any of the embodiments described herein, the metabolic disorder is fatty liver disease. In any of the embodiments described herein, the metabolic disorder is hypercholesterolemia. In any of the embodiments described herein, the metabolic disorder is elevated liver enzymes (eg, ALT and/or AST).

Metabolic disorders/conditions associated with body fat distribution include type 2 diabetes mellitus, hyperlipidemia or dyslipidemia (high levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very-low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid fractions). or altered circulating levels), obesity (particularly abdominal obesity), lipodystrophy (eg, inability to accumulate fat in local fat depots (partial lipodystrophy) or throughout the body (lipotrophy)), fasting or glucose or insulin Insulin resistance or higher or altered insulin levels, liver fat deposition or fatty liver disease and their complications (eg, cirrhosis, fibrosis or inflammation of the liver), high, elevated or altered liver enzyme levels or liver damage, inflammation or fat during challenge Deposits are not limited to other markers, high blood pressure and/or hypertension, hyperglycemia or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions and complications of each of the foregoing conditions.

In any of the embodiments described herein, the cardiovascular disease is cardiomyopathy, heart failure or hypertension. In any of the embodiments described herein, the cardiovascular disease is cardiomyopathy. In any of the embodiments described herein, the cardiovascular disease is heart failure. In any of the embodiments described herein, the cardiovascular disease is hypertension.

The present disclosure provides methods of treating a subject having or at risk of developing a metabolic disorder comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing type 2 diabetes comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing obesity comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing elevated triglyceride levels comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing NASH comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing lipodystrophy comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing liver inflammation comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing fatty liver disease comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing hypercholesterolemia comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing elevated liver enzymes (eg, ALT and/or AST) comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing a cardiovascular disease comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing cardiomyopathy, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing heart failure comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject having or at risk of developing high blood pressure comprising administering to the subject an INHBE inhibitor.

In some embodiments, an INHBE inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA) and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of INHBE mRNA. In some embodiments, the antisense RNA, siRNA or shRNA hybridizes to a sequence within an INHBE genomic nucleic acid molecule or mRNA molecule and reduces expression of an INHBE polypeptide in a cell of a subject. In some embodiments, an INHBE inhibitor comprises an antisense RNA that hybridizes to an INHBE genomic nucleic acid molecule or mRNA molecule and reduces expression of an INHBE polypeptide in a cell of a subject. In some embodiments, an INHBE inhibitor comprises an siRNA that hybridizes to an INHBE genomic nucleic acid molecule or mRNA molecule and reduces expression of an INHBE polypeptide in a cell of a subject. In some embodiments, an INHBE inhibitor comprises a shRNA that hybridizes to an INHBE genomic nucleic acid molecule or mRNA molecule and reduces expression of an INHBE polypeptide in a cell of a subject.

In some embodiments, the antisense nucleic acid molecule comprises or consists of the nucleotide sequences shown in Table 1.

Table 1

In some embodiments, the antisense nucleic acid molecule comprises or consists of the nucleotide sequences shown in Table 2.

Table 2

In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences shown in Table 3 (sense and antisense strands).

Table 3

In some embodiments, a siRNA molecule comprises or consists of the nucleotide sequences shown in Table 4 (sense and antisense strands).

Table 4

Inhibitory nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. An inhibitory nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence, such as in a vector or heterologous label. For example, an inhibitory nucleic acid molecule disclosed herein may exist within a vector or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. An inhibitory nucleic acid molecule may also be linked or fused to a heterologous label. A label may be directly detectable (eg, a fluorophore) or indirectly detectable (eg, a hapten, enzyme, or fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels and fluorescent labels. Labels include, for example, chemiluminescent materials; metal-containing materials; or an enzyme, wherein an enzyme-dependent secondary generation of the signal occurs. The term "label" can also refer to a "tag" or hapten capable of selectively binding to a conjugated molecule such that it is used to generate a detectable signal when the conjugated molecule is subsequently added with a substrate. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorescent substrate. Exemplary labels that can be used as tags to facilitate purification include myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags or Fc of immunoglobulins. Including but not limited to parts. A number of labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

The disclosed inhibitory nucleic acid molecules may include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutions. Such nucleotides include nucleotides that contain modified bases, sugar or phosphate groups or contain unnatural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorophore-labeled nucleotides.

An inhibitory nucleic acid molecule disclosed herein may also include one or more nucleotide analogs or substitutions. Nucleotide analogues are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to base moieties include, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenine, as well as natural and synthetic modifications of A, C, G, and T/U. -9-yl, but is not limited to different purine or pyrimidine bases. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, adenine and guanine. 2-propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-haluracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo (e.g., 5 -bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaguanine but is not limited to azaadenine, 3-deazaguanine and 3-deazaadenine.

Nucleotide analogs may also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C _1-10 alkyl or C _2-10 alkenyl, and C _2-10 alkynyl. Exemplary 2' sugar modifications are also -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n -ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , wherein n and m are independently 1 to about 10. Other modifications at the 2' position include C _1-10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. Similar modifications can be made at other positions of the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Modified sugars may also include those containing modifications at the bridging ring oxygens, such as CH ₂ and S. A nucleotide sugar analogue may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

Nucleotide analogs can also be modified at the phosphate moiety. The modified phosphate moiety is such that the linkage between two nucleotides is phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, 3'-alkylene phosphonate and methyl and other alkyl phosphonates, including chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thi include, but are not limited to, onoalkylphosphonates, thionoalkylphosphotriesters, and those that may be modified to contain boranophosphates. This phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, where the linkage is 3'-5' to 5'-3' or 2'-5' may contain an inverted polarity, such as from to 5'-2'. Various salts, mixed salts and free acid forms are also included. Nucleotide substitutions also include peptide nucleic acids (PNAs).

In some embodiments, the antisense nucleic acid molecule is a gapmer, such that the first 1 to 7 nucleotides at the 5' and 3' ends each have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides at the 5' and 3' ends each have a 2'-MOE modification. In some embodiments, the first 1 to 7 nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first 5 nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the backbone linkages between each nucleotide are phosphorothioate linkages.

In some embodiments, siRNA molecules have terminal modifications. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, non-hydrolyzable 5'-phosphate analogues such as 5'-(E)-vinyl-phosphonate are used.

In some embodiments, siRNA molecules have backbone modifications. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to improve the stability and in vivo bioavailability of siRNAs. The non-ester groups (—OH, =O) of the phosphodiester linkage can be replaced with sulfur, boron, or acetate to provide phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, replacing the phosphodiester group with a phosphotriester can remove the negative charge to promote cellular uptake and retention of siRNA in serum components. In some embodiments, siRNA molecules have sugar modifications. In some embodiments, the sugar deprotonates (a reaction catalyzed by exo- and endonucleases) so that the 2'-hydroxyl can act as a nucleophile and attack adjacent phosphorus at the phosphodiester bond. Such alternatives include 2'-0-methyl, 2'-0-methoxyethyl and 2'-fluoro modifications.

In some embodiments, siRNA molecules have base modifications. In some embodiments, bases may be substituted with modified bases such as pseudouridine, 5′-methylcytidine, N6-methyladenosine, inosine and N7-methylguanosine.

In some embodiments, siRNA molecules are conjugated to lipids. Lipids can be conjugated to the 5' or 3' ends of siRNAs to allow them to associate with serum lipoproteins, thereby improving bioavailability in vivo. Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitate and tocopherol.

In some embodiments, a representative siRNA has the formula:

Sense: mN*mN*/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/

i2FN/*mN*/32FN/

Antisense: /52FN/*/i2FN/*mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/

i2FN/mN/i2FN/mN*N*N

Where: “N” is a base; “2F” is the 2′-F variant; "m" is a 2'-O-methyl modification, "I" is an internal base; "*" is a phosphorothioate backbone linkage.

The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, a vector comprises any one or more inhibitory nucleic acid molecules disclosed herein and a heterologous nucleic acid. Vectors can be viral or non-viral vectors capable of transporting nucleic acid molecules. In some embodiments, a vector is a plasmid or cosmid (eg, circular double-stranded DNA to which additional DNA segments may be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Expression vectors include plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YAC), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.

The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition includes a carrier and/or excipient. Examples of carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates and lipid microtubules, but Not limited to this. Carriers may include buffered salt solutions such as PBS, HBSS, and the like.

In some embodiments, an INHBE inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in a DNA-binding protein or recognition sequence(s) that bind to a recognition sequence within an INHBE genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the INHBE gene or within a regulatory region that affects expression of the gene. The recognition sequence of a DNA-binding protein or nuclease agent may be located in an intron, exon, promoter, enhancer, regulatory region or any non-protein coding region. The recognition sequence may include or proximal to the initiation codon of the INHBE gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500 or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence that includes or proximal to the initiation codon. As another example, two nuclease preparations can be used, one targeting the nuclease recognition sequence containing or proximal to the start codon, and the other targeting the nuclease recognition sequence containing or proximal to the stop codon; , wherein cleavage by the nuclease agent can result in deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break into the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

Nuclease agents and DNA-binding proteins suitable for use herein include zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs). ), or Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. The length of the recognition sequence may vary, e.g. For example, a recognition sequence of about 30-36 bp for a zinc finger protein or ZFN pair, about 15-18 bp for each ZFN, about 36 bp for a TALE protein or TALEN, and about 20 bp for a CRISPR/Cas guide RNA. include

In some embodiments, the CRISPR/Cas system can be used to modify an INHBE genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein may use the CRISPR-Cas system by utilizing CRISPR complexes (including guide RNAs (gRNAs) complexed with Cas proteins) for site-directed cleavage of INHBE nucleic acid molecules.

A Cas protein usually contains at least one RNA recognition or binding domain capable of interacting with a gRNA. Cas proteins may also include nuclease domains (eg, DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains and other domains. Suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type Cpf1 protein (eg FnCpf1). The Cas protein can have full cleavage activity to create double-strand breaks in INHBE genomic nucleic acid molecules or it can be a nickase that creates single-strand breaks in INHBE genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modifications thereof Including but not limited to version. In some embodiments, a Cas system, such as Cas12a, can have multiple gRNAs encoded by a single crRNA. A Cas protein can also be operably linked to a heterologous polypeptide as a fusion protein. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain or a transcriptional repression domain. The Cas protein may be provided in any form. For example, the Cas protein may be provided in protein form, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule encoding the Cas protein, such as RNA or DNA.

In some embodiments, targeted genetic modification of an INHBE genomic nucleic acid molecule can be produced by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus within the INHBE genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1. The gRNA recognition sequence may include or proximate to the start codon of the INHBE genomic nucleic acid molecule or the stop codon of the INHBE genomic nucleic acid molecule. For example, a gRNA recognition sequence can be from about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon or stop codon. can be located

The gRNA recognition sequence within the target genomic locus of the INHBE genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The standard PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can carry Cas9 anywhere in the genome for gene editing, but editing cannot occur at sites other than where Cas9 recognizes the PAM. Also, 5'-NGA-3' can be a highly efficient non-standard PAM for human cells. Typically, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. PAMs can be flanked by gRNA recognition sequences. In some embodiments, a gRNA recognition sequence may be flanked at the 3' end by a PAM. In some embodiments, a gRNA recognition sequence may be flanked by a PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. As such, the PAM sequence of the complementary strand would be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

gRNA is an RNA molecule that binds to the Cas protein and targets the Cas protein to a specific location within the INHBE genomic nucleic acid molecule. An exemplary gRNA is a gRNA effective to direct a Cas enzyme to bind to or cleave an INHBE genomic nucleic acid molecule, wherein the gRNA comprises a DNA-targeting segment that hybridizes to a gRNA recognition sequence within the INHBE genomic nucleic acid molecule. An exemplary gRNA includes a DNA-targeting segment that hybridizes to a gRNA recognition sequence present in an INHBE genomic nucleic acid molecule that includes or is proximal to a start codon or a stop codon. For example, the gRNA may be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400 from the start codon. , about 500 or about 1,000 nucleotides or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200 of the stop codon , can be selected to hybridize to a gRNA recognition sequence located from about 300, about 400, about 500, or about 1,000 nucleotides. A suitable gRNA may comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, a gRNA can include 20 nucleotides.

Examples of suitable gRNA recognition sequences located within the human INHBE reference gene are set forth in Table 5 as SEQ ID NOs: 9-27.

Table 5: Guide RNA recognition sequences close to INHBE variant(s)

The Cas protein and gRNA form a complex and the Cas protein cleaves the target INHBE genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site within or outside the nucleic acid sequence present in the target INHBE genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA will bind. For example, formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) results in a single- or double-stranded nucleic acid sequence present in an INHBE genomic nucleic acid molecule to which the DNA-targeting segment of the gRNA will bind. cleavage within or near (eg, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs).

Such methods can generate, for example, INHBE genomic nucleic acid molecules in which the region of SEQ ID NO: 1 is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell may be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus of the INHBE genomic nucleic acid molecule. Cleavage by the Cas protein by contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes to a second gRNA recognition sequence) can generate two or more double-stranded breaks or two or more single-stranded breaks. can

The methods and compositions disclosed herein may utilize an exogenous donor sequence (e.g., a targeting vector or repair template) to modify the INHBE gene without cleavage of the INHBE gene or after cleavage of the INHBE gene with a nuclease agent. An exogenous donor sequence refers to any nucleic acid or vector that contains the necessary elements to allow site-specific recombination with a target sequence. The use of exogenous donor sequences in combination with nuclease agents can facilitate homology-directed repair, resulting in more precise modifications within the INHBE gene.

In this method, a nuclease agent cuts the INHBE gene to create single-stranded breaks (nicks) or double-stranded breaks, and the exogenous donor sequence is Non-homologous End Joining (NHEJ)-mediated Recombination of the INHBE gene through ligation or homology-directed repair events. Optionally, repair using an exogenous donor sequence removes or destroys the nuclease cleavage site so that the targeted allele cannot be retargeted by the nuclease agent.

The exogenous donor sequence may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and may be linear or circular. For example, the exogenous donor sequence can be a single-stranded oligodeoxynucleotide (ssODN) (Yoshimi et al., Nat. Commun., 2016, 7, 10431). Exemplary exogenous donor sequences are from about 50 nucleotides to about 5 kb in length, from about 50 nucleotides to about 3 kb in length, or from about 50 to about 1,000 nucleotides in length. Another exemplary exogenous donor sequence is about 40 to about 200 nucleotides in length. For example, the exogenous donor sequence may be about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, About 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, or about 190 to about 200 It can be nucleotides in length. Alternatively, the exogenous donor sequence is about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides in length. Alternatively, the exogenous donor sequence is about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, about 3 kb to 3.5 kb, about 3.5 kb. to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb in length. Alternatively, the exogenous donor sequence is, for example, 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides. , 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides or 50 nucleotides in length.

In some examples, the exogenous donor sequence is an ssODN that is about 80 nucleotides in length and about 200 nucleotides in length (eg, about 120 nucleotides in length). In another example, the exogenous donor sequence is an ssODN of about 80 nucleotides and about 3 kb in length. Such ssODNs may have, for example, homology arms that are about 40 nucleotides and about 60 nucleotides in length, respectively. Such ssODNs may also have homology arms that are, for example, about 30 nucleotides and 100 nucleotides in length, respectively. The homology arms can be symmetrical (e.g., each 40 nucleotides in length or each 60 nucleotides in length) or asymmetrical (e.g., one homology arm 36 nucleotides in length and 91 nucleotides in length). one homology arm).

The exogenous donor sequence may include modifications or sequences that provide additional desirable characteristics (eg, modified or modulated stability; tracking or detection using fluorescent labels; binding sites for proteins or protein complexes, etc.). The exogenous donor sequence may include one or more fluorescent labels, purification tags, epitope tags, or combinations thereof. For example, the exogenous donor sequence may include one or more fluorescent labels (eg, fluorescent proteins or other fluorophores or dyes), such as at least 1, at least 2, at least 3, at least 4 or at least 5 fluorescent labels. can Exemplary fluorescent labels include fluorescein (eg, 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-(and-6)- carboxytetramethylrhodamine (TAMRA), and fluorophores such as Cy7. A wide range of fluorescent dyes are commercially available for labeling oligonucleotides (eg, available from Integrated DNA Technologies). Such a fluorescent label (eg, an internal fluorescent label) can be used, for example, to detect an exogenous donor sequence directly integrated into a truncated INHBE gene having an overhanging end compatible with the end of the exogenous donor sequence. The label or tag may be at the 5' end, at the 3' end or within an exogenous donor sequence. For example, an exogenous donor sequence can be conjugated at the 5' end with an IR700 fluorophore ( ^5'IRDYE® 700) from Integrated DNA Technologies.

The exogenous donor sequence may also include a nucleic acid insert comprising a DNA segment to be integrated into the INHBE gene. Integration of a nucleic acid insert in an INHBE gene can result in the addition of a nucleic acid sequence of interest in the INHBE gene, deletion of a nucleic acid sequence of interest in the INHBE gene, or replacement (ie, deletion and insertion) of a nucleic acid sequence of interest in the INHBE gene. Some exogenous donor sequences are designed for insertion of nucleic acid inserts into the INHBE gene without any corresponding deletion in the INHBE gene. Other exogenous donor sequences are designed to delete the nucleic acid sequence of interest from the INHBE gene without the corresponding insertion of a nucleic acid insert. Another exogenous donor sequence is designed to delete a nucleic acid sequence of interest from the INHBE gene and replace it with a nucleic acid insert.

Nucleic acid inserts or corresponding nucleic acids within the INHBE gene that are deleted and/or replaced can be of various lengths. Exemplary nucleic acid inserts or corresponding nucleic acids within the INHBE gene that are deleted and/or replaced are from about 1 nucleotide to about 5 kb in length or from about 1 nucleotide to about 1,000 nucleotides in length. For example, a nucleic acid insert or corresponding nucleic acid in an INHBE gene that is deleted and/or replaced can be from about 1 to about 10, from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, About 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, or about 190 to about 200 nucleotides in length. Likewise, nucleic acid inserts or corresponding nucleic acids within the INHBE gene that are deleted and/or replaced can be from about 1 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides in length. Likewise, nucleic acid inserts or corresponding nucleic acids within the INHBE gene that are deleted and/or replaced may be about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, It may be about 3 kb to about 3.5 kb, about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb in length.

A nucleic acid insert may include genomic DNA or any other type of DNA. For example, a nucleic acid insert can include cDNA.

A nucleic acid insert may include sequences homologous to all or part of an INHBE gene (eg, part of a gene encoding a specific motif or region of an INHBE protein). For example, the nucleic acid insert comprises one or more point mutations (eg, 1, 2, 3, 4, 5 or more) or one or more nucleotide insertions or deletions compared to the sequence targeted for replacement in the INHBE gene. sequence may be included. Nucleic acid inserts or corresponding nucleic acids within the INHBE gene that are deleted and/or replaced include coding regions such as exons; non-coding regions such as introns, untranslated regions or regulatory regions (eg, promoters, enhancers or transcriptional repressor-binding elements); or a combination thereof.

Nucleic acid inserts may also include conditional alleles. The conditional allele may be a polyfunctional allele as described in US 2011/0104799. For example, a conditional allele may have: a) an operational sequence in sense orientation relative to the transcription of the target gene; b) a drug selection cassette (DSC) in sense or antisense orientation; c) a nucleotide sequence of interest (NSI) in antisense orientation; and d) conditional inversion modules in reverse orientation (COIN using exon-splitting introns and reversible gene-trap-like modules) (US 2011/0104799). The conditional allele i) lacks the working sequence and DSC; and ii) a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele comprising NSI in sense orientation and COIN in antisense orientation (US 2011/0104799).

A nucleic acid insert may also include a polynucleotide encoding a selectable marker. Alternatively, the nucleic acid insert may lack a polynucleotide encoding a selectable marker. A selection marker may be contained in a selection cassette. Optionally, the selection cassette may be a self-deleting cassette (US 8,697,851 and US 2013/0312129). As an example, a self-deleting cassette contains a Cre gene (comprising two exons encoding Cre recombinase separated by an intron) operably linked to the mouse Prml promoter and a neomycin resistance gene operably linked to the human ubiquitin promoter. can include Exemplary selection markers include neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin-N-acetyltransferase (puro ^r ), blasticidin S deaminase (bsr ^r ), xanthine/guanine phosphoribosyl transferase (gpt) or herpes simplex virus thymidine kinase (HSV-k) or combinations thereof. A polynucleotide encoding a selectable marker can be operably linked to a promoter that is active in the cell to be targeted. Examples of promoters are described elsewhere herein.

A nucleic acid insert may also include a reporter gene. Exemplary reporter genes include luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP). ), Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Seru including those encoding Cerulean, T-sapphire, alkaline phosphatase. Such a reporter gene can be operably linked to a promoter that is active in the cell to be targeted. Examples of promoters are described elsewhere herein.

A nucleic acid insert may also include one or more expression cassettes or deletion cassettes. A given cassette may contain one or more nucleotide sequences of interest, a polynucleotide encoding a selectable marker, and a reporter gene, along with various regulatory elements that affect expression. Examples of selectable markers and reporter genes that can be included are discussed in detail elsewhere herein.

Nucleic acid inserts may include nucleic acids flanked by site-specific recombination target sequences. Alternatively, the nucleic acid insert may include one or more site-specific recombination target sequences. Although the entire nucleic acid insert can be flanked by such site-specific recombination target sequences, any region or individual polynucleotide of interest within the nucleic acid insert can also be flanked by such sites. Site-specific recombination target sequences that may flank the nucleic acid insert or any polynucleotide of interest in the nucleic acid insert include, for example, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, or combinations thereof. In some instances, the site-specific recombination site is flanked by a polynucleotide encoding a selectable marker and/or reporter gene contained within the nucleic acid insert. After integrating the nucleic acid insert in the INHBE gene, the sequence between the site-specific recombination sites can be removed. Optionally, two exogenous donor sequences can be used, each with a nucleic acid insert comprising a site-specific recombination site. Exogenous donor sequences can target the 5' and 3' regions flanking the nucleic acid of interest. After integration of two nucleic acid inserts into a target genomic locus, the nucleic acid of interest between the two inserted site-specific recombination sites can be removed.

The nucleic acid insert may also contain one or more restriction sites for restriction endonucleases (ie, restriction enzymes), including type I, type II, type III and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sequences, but generally cleave at variable positions in the nuclease binding site, which can be hundreds of base pairs from the cleavage site (recognition sequence). In type II systems, restriction activity is independent of methylase activity, and cleavage usually occurs at specific sites within or near the binding site. Most type II enzymes cleave palindromic sequences, but type IIa enzymes recognize non-palindromic sequences and cleave outside the recognition sequence, type IIb enzymes cut sequences twice at two positions outside the recognition sequence, and type IIs The enzyme recognizes the asymmetric recognition sequence and cleave one side at a defined distance of about 1-20 nucleotides from the recognition sequence. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, eg in the REBASE database (webpage at rebase.neb.com; Roberts et al., Nucleic Acids Res., 2003, 31, 418-420; Roberts et al., Nucleic Acids Res., 2003, 31, 1805-1812; and Belfort et al., in Mobile DNA II, 2002, pp. 761-783, Eds. Craigie et al., (ASM Press, Washington, DC)).

Some exogenous donor sequences are located at the 5' end and/or 3' end complementary to one or more overhangs created by nuclease-mediated or Cas-protein-mediated cleavage at the target genomic locus (eg, in the INHBE gene). It has short single-stranded regions. These overhangs are also referred to as the 5' and 3' homology arms. For example, some exogenous donor sequences are located at the 5' end and/or 3' end complementary to one or more overhangs created by Cas-protein-mediated cleavage at the 5' and/or 3' target sequence at the target genomic locus. It has short single-stranded regions. Some of these exogenous donor sequences have regions complementary only to the 5' end or only to the 3' end. For example, some such exogenous donor sequences are complementary only at the 5' end complementary to overhangs created at the 5' target sequence at the target genomic locus or at the 3' end complementary to overhangs created at the 3' target sequence at the target genomic locus. has a complementary region only in Other such exogenous donor sequences have complementary regions at both the 5' and 3' ends. For example, other such exogenous donor sequences are complementary to complementary regions at both the 5' and 3' ends, eg, first and second overhangs, respectively, generated by Cas-mediated cleavage at the target genomic locus. For example, if the exogenous donor sequence is double-stranded, the single-stranded complementary region extends from the 5' end of the top strand of the donor sequence and the 5' end of the bottom strand of the donor sequence to form a 5' overhang at each end. can create Alternatively, a single-stranded complementary region can be extended from the 3' end of the top strand of the donor sequence and the 3' end of the bottom strand of the template to create a 3' overhang.

The complementary region may be of any length sufficient to facilitate ligation between the exogenous donor sequence and the INHBE gene. Exemplary complementary regions are about 1 to about 5 nucleotides in length, about 1 to about 25 nucleotides in length, or about 5 to about 150 nucleotides in length. For example, the complementary region is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Alternatively, the complementary region may be from about 5 to about 10, from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 60, About 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150 nucleotides in length, or more.

These complementary regions may be complementary to the overhangs created by the two pairs of nickases. Two staggered-end double-strand breaks consist of first and second nickases that cut opposite strands of DNA to create a first double-strand break, and opposite strands of DNA to create a second double-strand break. can be produced using third and fourth nickases that cleave For example, the Cas protein can be used to nick the first, second, third and fourth guide RNA recognition sequences corresponding to the first, second, third and fourth guide RNAs. The first and second guide RNA recognition sequences are such that the nicks generated by the first and second nickases on the first and second strands of DNA are double-stranded breaks (i.e., the first cleavage site is nicks in the RNA recognition sequence) to create a first cleavage site. Similarly, the third and fourth guide RNA recognition sequences are such that the nicks generated by the third and fourth nickases on the first and second strands of DNA are double-stranded breaks (i.e., the second cleavage site is the third and fourth nicks). nicks in the guide RNA recognition sequence) can be positioned to create a second cleavage site. Preferably, the nicks in the first and second guide RNA recognition sequences and/or the third and fourth guide RNA recognition sequences may be off-set nicks that create overhangs. The offset window can be, for example, at least about 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp or more (Ran et al ., Cell , 2013 , 154, 1380-1389; Mali et al . , Nat. Biotech., 2013, 31, 833-838; and Shen et al., Nat. Methods, 2014, 11, 399-404). In such case, the double-stranded exogenous donor sequence is single-stranded complementary to the overhangs created by the nicks in the first and second guide RNA recognition sequences and to the overhangs created by the nicks in the third and fourth guide RNA recognition sequences. It can be designed as a complementary region. Such exogenous donor sequences can be inserted by non-homologous-end-joining-mediated ligation.

Some exogenous donor sequences (ie, targeting vectors) contain homology arms. If the exogenous donor sequence also includes a nucleic acid insert, homology arms may flank the nucleic acid insert. For ease of reference, homology arms are referred to herein as 5' and 3' (ie, upstream and downstream) homology arms. The term relates to the position of homology arms relative to nucleic acid inserts within an exogenous donor sequence. The 5' and 3' homology arms correspond to regions within the INHBE gene referred to herein as "5' target sequence" and "3' target sequence", respectively.

A homology arm and a target sequence are “corresponding” or “corresponding” to each other when the two regions share a sufficient level of sequence identity with each other to act as substrates for a homologous recombination reaction. The term “homologous” includes DNA sequences that are identical or share sequence identity with a corresponding sequence. Sequence identity between a given target sequence and the corresponding homology arms found in the exogenous donor sequence can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arms of the exogenous donor sequence (or fragment thereof) and the target sequence (or fragment thereof) is at least 50%, 55%, 60%, 65%, such that the sequences undergo homologous recombination. %, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Moreover, the corresponding region of homology between the homology arm and the corresponding target sequence may be of any length sufficient to promote homologous recombination. Exemplary homology arms are from about 25 nucleotides to about 2.5 kb in length, or from about 25 nucleotides to about 1.5 kb in length, or from about 25 nucleotides to about 500 nucleotides in length. For example, a given homology arm (or each homology arm) and/or the corresponding target sequence may be between about 25 and 50 so that the homology arm has sufficient homology to undergo homologous recombination with the corresponding target sequence in the INHBE gene. About 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 350, about 350 to about 400, about 400 to about 450 , or about 450 to about 500 nucleotides in length. Alternatively, a given homology arm (or each homology arm) and/or corresponding target sequence may be between about 0.5 kb and about 1 kb, between about 1 kb and about 1.5 kb, between about 1.5 kb and about 2 kb, or about 2 kb. kb to about 2.5 kb in length. For example, the homology arms may each be about 750 nucleotides in length. Homologous arms can be symmetrical (each approximately the same size in length) or asymmetrical (one longer than the other).

A homology arm may correspond to a cell-specific locus (eg, a target locus). Alternatively, they may correspond to regions of heterologous or exogenous segments of DNA integrated into the genome of the cell, including, for example, transgenes, expression cassettes, or heterologous or exogenous regions of DNA. Alternatively, the homologous arm of the targeting vector may correspond to a region of a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other engineered region contained in a suitable host cell. In addition, the homology arms of the targeting vector may correspond to or be derived from regions of a BAC library, cosmid library, or P1 phage library, or may be derived from synthetic DNA.

When the nuclease agent is used in combination with an exogenous donor sequence, the 5' and 3' target sequences are preferably identical to the target sequence upon single-strand break (nick) or double-strand break at the nuclease cleavage site. It is located in close enough proximity to the nuclease cleavage site to facilitate the occurrence of homologous recombination events between homologous arms. The term "nuclease cleavage site" includes DNA sequences where nicks or double-strand breaks are created by nuclease agents (eg, Cas9 proteins complexed with guide RNAs). 5' of an exogenous donor sequence, if the distance is such that single-strand breaks or double-strand breaks at the nuclease cleavage site promote the occurrence of homologous recombination events between the 5' and 3' target sequences and the homology arms. and the target sequence in the INHBE gene corresponding to the 3' homology arm is "located in close proximity" to the nuclease cleavage site. Thus, a target sequence corresponding to the 5' and/or 3' homology arms of an exogenous donor sequence may, for example, be within at least 1 nucleotide of a given nuclease cleavage site or within at least 10 nucleotides of a given nuclease cleavage site. nucleotide to about 1,000 nucleotides. By way of example, the nuclease cleavage site may be immediately adjacent to at least one or both of the target sequences.

The spatial relationship of the homology arms of the exogenous donor sequence and the target sequence corresponding to the nuclease cleavage site can vary. For example, the target sequence may be located 5' of the nuclease cleavage site, the target sequence may be located 3' of the nuclease cleavage site, or the target sequence may be located on the side of the nuclease cleavage site. can do.

Also provided are methods of treating and treating or preventing metabolic disorders in a subject having or at risk of developing the disease using the methods disclosed herein for modifying or altering the expression of an endogenous INHBE gene. Having a disease or using a method of reducing the expression of an INHBE mRNA transcript or providing a recombinant nucleic acid encoding an INHBE protein, providing mRNA encoding an INHBE protein, or providing an INHBE protein to a subject Methods of treating and treating or preventing metabolic disorders in subjects at risk of developing them are also provided. The method may include introducing (eg, in vivo or ex vivo) one or more nucleic acids or proteins into a subject, into the subject's liver, or into a cell (eg, liver cell) of the subject.

Also provided are methods of treating and treating or preventing cardiovascular disease in a subject having or at risk of cardiovascular disease using the methods disclosed herein for modifying or altering the expression of an endogenous INHBE gene. In addition, using a method of reducing the expression of an INHBE mRNA transcript or providing a recombinant nucleic acid encoding an INHBE protein and providing an mRNA encoding an INHBE protein or providing an INHBE protein to a subject having a cardiovascular disease Provided are methods of treating and treating or preventing cardiovascular disease in a subject at risk or at risk. The method may include introducing (eg, in vivo or ex vivo) one or more nucleic acids or proteins into a subject, into the subject's liver, or into a cell (eg, liver cell) of the subject.

Such methods may include genome editing or gene therapy. For example, an endogenous INHBE gene that does not encode a loss-of-function variant can be modified to include any loss-of-function variant described herein. As another example, an endogenous INHBE gene that does not encode a loss-of-function variant can be knocked out or inactivated. Likewise, endogenous INHBE genes that do not encode loss-of-function variants can be knocked out or inactivated, and INHBE genes comprising any one or any combination of INHBE loss-of-function variants described herein can be introduced and expressed. It can be. Similarly, endogenous INHBE genes that do not encode loss-of-function variants can be knocked out or inactivated, and recombinant DNA encoding any one or any combination of INHBE loss-of-function variants described herein can be introduced can be expressed, and mRNA encoding any one or any combination of INHBE loss-of-function variants (or fragments thereof) described herein can be introduced and expressed (eg, intracellular protein replacement therapy); or or cDNA encoding any one or any combination of INHBE loss-of-function variants (or fragments thereof) described herein may be introduced (eg, protein replacement therapy).

Other such methods include introducing and expressing a recombinant INHBE gene comprising any one or any combination of INHBE loss-of-function variants described herein (e.g., full INHBE variants or minigenes comprising variants); introducing and expressing a recombinant nucleic acid (e.g., DNA) encoding any one or any combination of INHBE loss-of-function variants or fragments thereof described herein; introducing and expressing one or more mRNAs encoding any one of the fragments or any combination thereof (eg, intracellular protein replacement therapy), or knocking out the endogenous INHBE gene that does not encode the loss-of-function variant. introducing any one or any combination of the INHBE loss-of-function variants described herein (eg, protein replacement therapy) without inactivating them or inactivating them.

An INHBE gene or minigene or DNA encoding any one or any combination of INHBE loss-of-function variants or fragments thereof described herein may be introduced and expressed in the form of an expression vector that does not modify the genome, which does not modify the INHBE It may be introduced in the form of a targeting vector to be genomically integrated into the locus, or introduced to be genomically integrated into a locus other than the INHBE locus, such as the safe harbor locus. A genomically integrated INHBE gene can be operably linked at the integration site to the INHBE promoter or another promoter, such as an endogenous promoter. A safe harbor locus is a chromosomal region where the transgene can be stably and reliably expressed in all tissues of interest without adversely affecting gene structure or expression. A safe harbor locus can, for example, have one or more or all of the following characteristics: a distance of at least 50 kb from the 5' end of any gene; a distance of 300 kb or greater from a cancer-related gene; a distance of 300 kb or greater from any microRNA; Outside gene transcription units and outside super-conserved regions. Examples of suitable safe harbor loci include the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) gene locus, and the human orthologue of the mouse ROSA26 locus.

INHBE protein isoforms or combinations of nucleic acids encoding INHBE protein isoforms that can be introduced and expressed include any one or any combination of protein or mRNA isoforms described herein. For example, an INHBE nucleic acid encoding isoform 1 (SEQ ID NO: 2) encoding any one or any combination of loss-of-function variants described herein (alone or in combination with other isoforms) is introduced. become or appear Exemplary sequences for each of these isoforms and transcripts are provided elsewhere herein. However, it is understood that within gene sequences and populations, mRNA sequences transcribed from such genes, and proteins translated from such mRNAs, may vary due to polymorphisms, such as single-nucleotide polymorphisms. The sequences provided herein for each transcript and isoform are exemplary sequences only. Other sequences are also possible.

In some embodiments, the method is not a carrier of any INHBE variant nucleic acid molecule described herein (or only a heterozygous carrier of any one or any combination of variant nucleic acid molecules described herein) and/or a metabolic disorder and/or a) a nuclease agent (or encoding nucleic acid) that binds to a nuclease recognition sequence in an INHBE gene, wherein the nuclease recognition sequence is described herein; comprises or is proximal to a position in one of the INHBE variant nucleic acid molecules; and b) a 5' homology arm that hybridizes to a target sequence 5' of a position of one of the INHBE variant nucleic acid molecules described herein, a 3' homology arm that hybridizes to a target sequence 3' of the same INHBE variant nucleic acid molecule, and 5' introducing into a subject or into liver cells of a subject an exogenous donor sequence comprising a nucleic acid insert comprising a homology arm and one or more variant nucleotides flanked by a 3' homology arm. The nuclease agent is capable of cleaving the INHBE gene in liver cells of a subject, and the exogenous donor sequence is capable of recombination with the INHBE gene in liver cells, wherein upon recombination of the exogenous donor sequence and the INHBE gene, a loss-of-function variant is identified. A nucleic acid insert of interest is introduced to replace the wild-type nucleotide. Examples of nuclease agents (eg, Cas9 proteins and guide RNAs) that can be used in these methods are disclosed elsewhere herein. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. Examples of exogenous donor sequences that can be used in these methods are disclosed elsewhere herein.

As another example, the method is not a carrier of any INHBE variant nucleic acid molecule described herein (or only a heterozygous carrier of any one or any combination of variant nucleic acid molecules described herein) and/or a metabolic disorder and/or or a 5' homology arm that hybridizes to a target sequence 5' of a position of one of the INHBE variant nucleic acid molecules described herein, a target of the same INHBE variant nucleic acid molecule, comprising treating a subject having or susceptible to developing a cardiovascular disease. introducing into a subject an exogenous donor sequence comprising a nucleic acid insert comprising a 3' homology arm that hybridizes to sequence 3' and one or more variant nucleotides flanking the 5' homology arm and the 3' homology arm; Including introducing into liver cells. The exogenous donor sequence is capable of recombination with the INHBE gene in liver cells, where upon recombination of the exogenous donor sequence with the INHBE gene, a nucleic acid insert encoding a loss-of-function variant is introduced to replace the wild-type nucleotide. Examples of exogenous donor sequences that can be used in these methods are disclosed elsewhere herein.

In some embodiments, the method is not a carrier of any INHBE variant nucleic acid molecule described herein (or only a heterozygous carrier of any one or any combination of variant nucleic acid molecules described herein) and/or a metabolic disorder and/or or treating a subject having or susceptible to developing a cardiovascular disease, wherein a) introducing into the subject a nuclease agent (or encoding nucleic acid) that binds to a nuclease recognition sequence in the INHBE gene or introducing into liver cells of the subject wherein the nuclease recognition sequence comprises or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the initiation codon for the INHBE gene . Nuclease agents can cleave and disrupt expression of the INHBE gene in liver cells in a subject. In some embodiments, the method is not a carrier of any INHBE variant nucleic acid molecule described herein (or only a heterozygous carrier of any one or any combination of variant nucleic acid molecules described herein) and/or a metabolic disorder and/or or treating a subject having or susceptible to having a cardiovascular disease, wherein a) a nuclease agent (or encoding nucleic acid) that binds to a nuclease recognition sequence in the INHBE gene, wherein the nuclease recognition sequence is in the INHBE gene comprises the initiation codon for or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the initiation codon or is selected from SEQ ID NOs: 1-7; and b) introducing into a subject or into liver cells of a subject an expression vector comprising a recombinant INHBE gene comprising any one or any combination of loss-of-function variants described herein. Expression vectors may be non-genomically integrated. Alternatively, a targeting vector comprising a recombinant INHBE gene comprising any one or any combination of loss-of-function variants described herein (ie, an exogenous donor sequence) may be introduced. The nuclease agent can cleave and disrupt expression of the INHBE gene in liver cells in a subject, and the expression vector can express the recombinant INHBE gene in liver cells in a subject. Alternatively, the genomically integrated recombinant INHBE gene can be expressed in liver cells in a subject. Examples of nuclease agents (eg, nuclease-active Cas9 proteins and guide RNAs) that can be used in these methods are disclosed elsewhere herein. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. Step b) alternatively comprises an INHBE protein that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE isoform or fragment thereof described herein. introducing an expression vector or targeting vector comprising an encoding nucleic acid (eg, DNA) and comprising any one or any combination of INHBE variant nucleic acid molecules described herein. Similarly, step b) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE mRNA isoform or fragment thereof described herein and introducing an mRNA encoding an INHBE protein comprising any one or any combination of the described INHBE variant nucleic acid molecules. Similarly, step b) comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any INHBE protein isoform or fragment thereof described herein; It may alternatively involve introducing a protein comprising any one or any combination of loss-of-function variant polypeptides described herein.

In some embodiments, the second nuclease agent is also introduced into the subject or liver cells of the subject, wherein the second nuclease agent binds to a second nuclease recognition sequence in the INHBE gene, wherein the second nuclease agent The first recognition sequence comprises a stop codon for the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the stop codon, wherein the nuclease agent is cleavage of the INHBE gene within both the first nuclease recognition sequence and the second nuclease recognition sequence in a liver cell, wherein the liver cell comprises a deletion between the first nuclease recognition sequence and the second nuclease recognition sequence; transformed to do For example, the second nuclease agent can be a Cas9 protein and guide RNA. Suitable guide RNAs and guide RNA recognition sequences proximal to the stop codon are disclosed elsewhere herein.

Such methods are not carriers of any INHBE variant nucleic acid molecules described herein (or only heterozygous carriers of any one or any combination of INHBE variant nucleic acid molecules described herein), and metabolic disorders and/or cardiovascular disease. a) introducing a DNA-binding protein (or encoding nucleic acid) that binds to a DNA-binding protein recognition sequence in the INHBE gene into the subject or into liver cells of the subject; wherein the DNA-binding protein recognition sequence comprises or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the initiation codon for the INHBE gene. The DNA-binding protein can alter (eg, decrease) the expression of the INHBE gene in liver cells in a subject. Such methods are not carriers of any INHBE variant nucleic acid molecules described herein (or only heterozygous carriers of any one or any combination of INHBE variant nucleic acid molecules described herein), and metabolic disorders and/or cardiovascular disease. a) a DNA-binding protein (or encoding nucleic acid) that binds to a DNA-binding protein recognition sequence in an INHBE gene, wherein the DNA-binding protein recognition sequence is disclosed for the INHBE gene; codon or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the initiation codon; and b) introducing into a subject or into liver cells of a subject an expression vector comprising a recombinant INHBE gene comprising any one or any combination of loss-of-function variants described herein. Expression vectors may be non-genomically integrated. Alternatively, a targeting vector comprising a recombinant INHBE gene comprising any one or any combination of INHBE variant nucleic acid molecules described herein (ie, an exogenous donor sequence) may be introduced. The DNA-binding protein can alter (eg, decrease) expression of the INHBE gene in liver cells in a subject, and the expression vector can express the recombinant INHBE gene in liver cells in the subject. Alternatively, the genomically integrated recombinant INHBE gene can be expressed in liver cells in a subject. Examples of DNA-binding proteins suitable for use in these methods are disclosed elsewhere herein. Such DNA-binding proteins (eg, Cas9 protein and guide RNA) can be fused to or operably linked to transcriptional repression domains. For example, the DNA-binding protein can be a catalytically inactive Cas9 protein fused to a transcriptional repression domain. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. step b) is alternatively at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE isoform or fragment thereof described herein introducing an expression vector or targeting vector comprising a nucleic acid (eg, DNA) encoding an INHBE protein comprising any one or any combination of the described INHBE variant nucleic acid molecules. Similarly, step b) is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE mRNA isoform or fragment thereof described herein and introducing an mRNA encoding an INHBE protein comprising any one or any combination of the described INHBE variant nucleic acid molecules. Likewise, step b) comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any INHBE protein isoform or fragment thereof described herein; It may alternatively include introducing a protein comprising any one or any combination of loss-of-function variant polypeptides described herein.

Such methods are not carriers of any INHBE variant nucleic acid molecules described herein (or only heterozygous carriers of any one or any combination of INHBE variant nucleic acid molecules described herein), and metabolic disorders and/or cardiovascular disease. comprising treating a subject having or susceptible to the disease, comprising introducing an expression vector into the subject or into liver cells of the subject, wherein the expression vector is any one or any combination of loss-of-function variants described herein. A recombinant INHBE gene comprising a, wherein the expression vector expresses the recombinant INHBE gene in liver cells of a subject. Expression vectors may be non-genomically integrated. Alternatively, a targeting vector comprising a recombinant INHBE gene comprising any one or any combination of INHBE variant nucleic acid molecules described herein (ie, an exogenous donor sequence) may be introduced. In methods where an expression vector is used, the expression vector is capable of expressing the recombinant INHBE gene in liver cells in a subject. Alternatively, in methods where the recombinant INHBE gene is genomically integrated, the recombinant INHBE gene can be expressed in liver cells in a subject. Such methods are alternatively at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE isoform or fragment thereof described herein and described herein. introducing an expression vector or targeting vector comprising a nucleic acid (eg, DNA) encoding an INHBE protein comprising any one or any combination of loss-of-function variants. Likewise, such methods are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any INHBE mRNA isoform or fragment thereof described herein and described herein. Introducing mRNA encoding an INHBE protein comprising any one or any combination of INHBE variant nucleic acid molecules. Likewise, such methods are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any INHBE protein isoform or fragment thereof described herein and have the functions described herein. -introducing a protein comprising any one or any combination of the missing variant polypeptides.

Expression vectors and recombinant INHBE genes suitable for use in any of the above methods are disclosed elsewhere herein. For example, a recombinant INHBE gene can be a full-length variant gene or an INHBE minigene in which one or more non-essential segments of the gene have been deleted relative to the corresponding wild-type INHBE gene. For example, a deleted segment may include one or more intronic sequences. An example of a complete INHBE gene is one that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 when optimally aligned with SEQ ID NO: 1.

In some embodiments, the method comprises altering cells (eg, liver cells) in a subject having or susceptible to developing a chronic liver disease. In some embodiments, the method comprises modifying cells (eg, cardiac cells) in a subject having or susceptible to developing a cardiovascular disease. In such methods, the nuclease agent and/or exogenous donor sequence and/or recombinant expression vector delay onset, reduce severity, inhibit further deterioration and/or ameliorate at least one sign or symptom of the disease being treated. It can be introduced into cells through administration with an effective regimen, which means the dosage, route of administration, and frequency of administration. The term "symptom" refers to subjective evidence of disease perceived by a subject, and "sign" refers to objective evidence of disease observed by a physician. When a subject already suffers from a disease, the therapy may be referred to as a therapeutically effective therapy. When a subject is at high risk of disease compared to the general population but does not yet experience symptoms, this therapy can be said to be prophylactically effective. In some cases, a therapeutic or prophylactic efficacy may be observed in an individual patient relative to a past control group or past experience with the same subject. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a preclinical or clinical trial in a population of treated subjects compared to a control population of untreated subjects.

As disclosed elsewhere herein, delivery may be by any suitable method. For example, the nuclease agent or exogenous donor sequence or recombinant expression vector can be used for vector delivery, viral delivery, particle-mediated delivery, nanoparticle-mediated delivery, liposome-mediated delivery, exosome-mediated delivery, lipid-mediated delivery, It can be delivered by lipid-nanoparticle mediated delivery, cell-penetrating-peptide-mediated delivery or implantable-device mediated delivery. Some specific examples include hydrodynamic delivery, virus-mediated delivery and lipid-nanoparticle-mediated delivery. Administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. For example, a specific example frequently used in protein replacement therapy is intravenous infusion. The frequency of administration and number of doses may vary depending on the half-life of the nuclease agent or exogenous donor sequence or recombinant expression vector, the condition of the subject, and the route of administration, among other factors. Pharmaceutical compositions for administration are preferably sterile, substantially isotonic, and prepared under GMP conditions. A pharmaceutical composition may be presented in unit dosage form (ie, a dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or adjuvants. Formulations depend on the route of administration chosen. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and is not substantially injurious to the recipient.

Other such methods include ex vivo methods in cells from a subject having or susceptible to developing chronic liver disease and/or cardiovascular disease. The cells with the target genetic modification can then be transplanted back into the subject.

In some embodiments, an INHBE inhibitor comprises a small molecule. In some embodiments, the INHBE inhibitor is any inhibitory nucleic acid molecule described herein. In some embodiments, an INHBE inhibitor comprises an antibody.

In some embodiments, the treatment method detects the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, or the presence of a corresponding INHBE polypeptide, or an INHBE polypeptide or nucleic acid (e.g., eg, RNA). As used throughout this disclosure, an "INHBE variant nucleic acid molecule" refers to any INHBE polypeptide encoding an INHBE polypeptide having partial loss-of-function, complete loss-of-function, predicted partial-loss of function, or predicted complete loss-of-function. of INHBE nucleic acid molecules (eg, genomic nucleic acid molecules, mRNA molecules or cDNA molecules).

The present disclosure also provides methods of treating a subject suffering from a metabolic disorder with a therapeutic agent that treats or inhibits the metabolic disorder. In some embodiments, a method comprises obtaining or obtaining a biological sample from a subject and performing or performing genotyping on the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule, such that the subject has an INHBE predicted function- and determining whether it has an INHBE variant nucleic acid molecule encoding the missing polypeptide. If the subject is on INHBE criteria, a therapeutic agent that treats or inhibits the metabolic disorder is administered to the subject at a standard dose or continues to be administered, and an INHBE inhibitor is administered to the subject. If the subject is heterozygous for the INHBE variant nucleic acid molecule, a therapeutic agent that treats or inhibits the metabolic disorder is administered or continues to be administered to the subject in an amount equal to or lower than the standard dose, and an INHBE inhibitor is administered to the subject. When the subject is homozygous for the INHBE variant nucleic acid molecule, a therapeutic agent that treats or inhibits the metabolic disorder is administered to the subject in an amount equal to or lower than the standard dose, or continues to be administered. The presence of a genotype with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing a metabolic disorder. In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In the case of a subject genotyped or determined to be INHBE criteria or heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, such subject may be treated with an INHBE inhibitor as described herein.

The present disclosure also provides methods of treating a subject suffering from cardiovascular disease with a therapeutic agent that treats or inhibits cardiovascular disease. In some embodiments, a method comprises obtaining or obtaining a biological sample from a subject and performing or performing genotyping on the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule, such that the subject is INHBE predicted-loss-of-function. and determining whether it has an INHBE variant nucleic acid molecule encoding the polypeptide. When the subject is on INHBE criteria, a therapeutic agent that treats or inhibits cardiovascular disease is administered to the subject at a standard dose or continues to be administered, and an INHBE inhibitor is administered to the subject. If the subject is heterozygous for the INHBE variant nucleic acid molecule, a therapeutic agent that treats or inhibits cardiovascular disease is administered or continues to be administered to the subject in an amount equal to or lower than the standard dose, and an INHBE inhibitor is administered to the subject. When a subject is homozygous for the INHBE variant nucleic acid molecule, a therapeutic agent that treats or inhibits cardiovascular disease is administered to the subject in an amount equal to or lower than the standard dose or continues to be administered. The presence of a genotype with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing cardiovascular disease. In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In the case of a subject who has been genotyped or has been determined to be INHBE criterion or heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, such subject can be treated with an INHBE inhibitor as described herein.

Detecting the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample from a subject and/or determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide Doing may be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from a subject.

In some embodiments, when the subject is on INHBE criteria, the subject is also administered a therapeutic agent that treats or inhibits the metabolic disorder at standard dosages. In some embodiments, where the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject also treats or inhibits a metabolic disorder at an amount equal to or less than a standard dose. receive treatment for

In some embodiments, when the subject is on INHBE criteria, the subject is also administered a therapeutic agent that treats or inhibits cardiovascular disease at standard dosages. In some embodiments, where the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject also treats or inhibits cardiovascular disease at an amount equal to or less than a standard dose. receive treatment for

In some embodiments, the method of treatment further comprises detecting the presence or absence of an INHBE predicted loss-of-function polypeptide in a biological sample from the subject. In some embodiments, if the subject does not have the INHBE predicted loss-of-function polypeptide, the subject is also administered a standard dosage of a therapeutic agent that treats or inhibits the metabolic disorder. In some embodiments, if the subject has an INHBE predicted loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than the standard dosage.

In some embodiments, the method of treatment further comprises detecting the presence or absence of an INHBE predicted loss-of-function polypeptide in a biological sample from the subject. In some embodiments, if the subject does not have the INHBE predicted loss-of-function polypeptide, the subject is also administered a standard dosage of a therapeutic agent that treats or inhibits cardiovascular disease. In some embodiments, if the subject has an INHBE predicted loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or less than the standard dosage.

The present disclosure also provides methods of treating a subject suffering from a metabolic disorder with a therapeutic agent that treats or inhibits the metabolic disorder. In some embodiments, the method comprises obtaining or obtaining a biological sample from a subject and performing or performing an assay on the biological sample to determine whether the subject has an INHBE predicted loss-of-function polypeptide, such that the subject has an INHBE predicted loss-of-function polypeptide. It includes determining whether or not to have If the subject does not have the INHBE predicted loss-of-function polypeptide, a therapeutic agent that treats or inhibits the metabolic disorder is administered or continues to be administered to the subject at a standard dose, and an INHBE inhibitor is administered to the subject. If the subject has an INHBE predicted loss-of-function polypeptide, a therapeutic agent that treats or inhibits the metabolic disorder is administered or continues to be administered to the subject in an amount equal to or less than a standard dose, and an INHBE inhibitor is administered to the subject. The presence of the INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing a metabolic disorder. In some embodiments, the subject has an INHBE predicted loss-of-function polypeptide. In some embodiments, the subject does not have an INHBE predicted loss-of-function polypeptide.

The present disclosure also provides methods of treating a subject suffering from cardiovascular disease with a therapeutic agent that treats or inhibits cardiovascular disease. In some embodiments, the method comprises obtaining or obtaining a biological sample from a subject and performing or performing an assay on the biological sample to determine whether the subject has an INHBE predicted loss-of-function polypeptide, such that the subject has an INHBE predicted loss-of-function polypeptide. It includes determining whether or not to have If the subject does not have the INHBE predicted loss-of-function polypeptide, a therapeutic agent that treats or inhibits the cardiovascular disease is administered or continues to be administered to the subject at a standard dose, and an INHBE inhibitor is administered to the subject. If the subject has an INHBE predicted loss-of-function polypeptide, a therapeutic agent that treats or inhibits the cardiovascular disease is administered to the subject at the same or less than standard dose or continues to be administered, and the INHBE inhibitor is administered to the subject. The presence of the INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing cardiovascular disease. In some embodiments, the subject has an INHBE predicted loss-of-function polypeptide. In some embodiments, the subject does not have an INHBE predicted loss-of-function polypeptide.

Detecting the presence or absence of an INHBE predicted loss-of-function polypeptide in a biological sample from a subject and/or determining whether a subject has an INHBE predicted loss-of-function polypeptide can be performed by any of the methods described herein. there is. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide may be present in a cell or blood sample obtained from the subject or other information about the subject previously generated from collection of a cell or blood sample from the subject or a biological relative of the subject. In any of these embodiments, a determination by quantification of the amount of INHBE polypeptide can be included as a determination of loss of function due to an effective absence or reduction in the amount of INHBE polypeptide. In any of these embodiments, detection, sequencing and/or quantification of INHBE DNA and RNA can serve as a method for determining loss-of-function or absence of INHBE.

Examples of therapeutic agents that treat or inhibit type 2 diabetes include metformin, insulin, sulfonylureas (eg glyburide, glipizide and glimepiride), meglitinides (eg repaglinide and nategli thiazolidinediones (e.g. rosiglitazone and pioglitazone), DPP-4 inhibitors (e.g. sitagliptin, saxagliptin, linagliptin), GLP-1 receptor agonists (e.g. ecgliptin) senataide, liraglutide, semaglutide), SGLT2 inhibitors (eg canagliflozin, dapagliflozin, empagliflozin). In some embodiments, the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, lira glutide, semaglutide, canagliflozin, dapagliflozin or empagliflozin. In some embodiments, the therapeutic agent is metformin. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is glyburide. In some embodiments, the therapeutic agent is glipizide. In some embodiments, the therapeutic agent is glimepiride. In some embodiments, the therapeutic agent is repaglinide. In some embodiments, the therapeutic agent is nateglinide. In some embodiments, the therapeutic agent is rosiglitazone. In some embodiments, the therapeutic agent is pioglitazone. In some embodiments, the therapeutic agent is sitagliptin. In some embodiments, the therapeutic agent is saxagliptin. In some embodiments, the therapeutic agent is linagliptin. In some embodiments, the therapeutic agent is exenatide. In some embodiments, the therapeutic agent is liraglutide. In some embodiments, the therapeutic agent is semaglutide. In some embodiments, the therapeutic agent is canagliflozin. In some embodiments, the therapeutic agent is dapagliflozin. In some embodiments, the therapeutic agent is empagliflozin.

Examples of therapeutic agents that treat or inhibit obesity include, but are not limited to, orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide. In some embodiments, the therapeutic agent is orlistat. In some embodiments, the therapeutic agent is phentermine. In some embodiments, the therapeutic agent is topiramate. In some embodiments, the therapeutic agent is bupropion. In some embodiments, the therapeutic agent is naltrexone. In some embodiments, the therapeutic agent is liraglutide.

Examples of therapeutic agents that treat or inhibit elevated triglycerides include statins (eg, rosuvastatin, simvastatin, and atorvastatin), fibrates (eg, fenofibrate, gemfibrozil, and fenofibric acid), nicotinic acid (eg, rosuvastatin, simvastatin, and atorvastatin). , niacin) and fatty acids (eg, omega-3 fatty acids). In some embodiments, the therapeutic agent is a statin.

Examples of therapeutic agents that treat or inhibit lipodystrophy include ^EGRIFTA® (tesamorelin), ^GLUCOPHAGE® (metformin), ^SCULPTRA® (poly-L-lactic acid), ^RADIESSE® (calcium hydroxyapatite), polymethylmethacrylate ( Examples include, but are not limited to, PMMA), ZYDERM ^® (bovine collagen), COSMODERM ^® (human collagen), silicone, glitazone, and hyaluronic acid. In some embodiments, the therapeutic agent that treats or inhibits lipodystrophy includes tesamorelin, metformin, poly-L-lactic acid, calcium hydroxyapatite, polymethylmethacrylate, bovine collagen, human collagen, silicone, and hyaluronic acid. Not limited to this.

Examples of therapeutics that treat or inhibit liver inflammation include, but are not limited to, hepatitis treatments and hepatitis vaccines.

Examples of therapeutic agents or procedures that treat or inhibit fatty liver disease include, but are not limited to, bariatric surgery and/or dietary intervention.

Examples of therapeutic agents that treat or inhibit hypercholesterolemia include statins (e.g., ^LIPITOR® (atorvastatin), ^LESCOL® (fluvastatin), lovastatin, ^LIVALO® (pitavastatin), ^PRAVACHOL® (pravastatin), ^CRESTOR® (rosuvastatin calcium), and ^ZOCOR® (simvastatin)); bile acid sequestrants (eg, ^PREVALITE® (cholestyramine), ^WELCHOL® (colesevelam) and ^COLESTID® (colestipol)); PCSK9 inhibitors (eg, ^PRALUENT® (alirocumab) and ^REPATHA® (evolocumab)); niacin (eg, niaspan and niacor); fibrates (eg fenofibrate and LOPID ^® (gemfibrozil)); and ATP citrate lyase (ACL) inhibitors (eg ^NEXLETOL® (Bempedoic)). In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is a statin (eg, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, and simvastatin); bile acid sequestrants (eg cholestyramine, colesevelam and colestipol); PCSK9 inhibitors (e.g. alirocumab and evolocumab; niacins (e.g. niaspan and niacor); fibrates (e.g. fenofibrate and gemfibrozil); and ACL inhibitors (e.g. In some embodiments, the therapeutic agent for treating or inhibiting hypercholesterolemia is alirocumab or evolocumab In some embodiments, the therapeutic agent for treating or inhibiting hypercholesterolemia is alirocumab In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is evolocumab.

Examples of therapeutic agents that treat or inhibit elevated liver enzymes (e.g., ALT and/or AST) include, but are not limited to, coffee, folic acid, potassium, vitamin B6, statins and fiber, or any combination thereof .

Examples of therapeutic agents that treat or inhibit NASH include OCALIVA ^® (obeticholic acid), pioglitazone or other glitazones, celonsertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E, arachidylamidocholanosic acid ( ARAMCHOL ^™ ), GS0976, emricasan, bolixivat, NGM282, GS9674, tropexor, MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, saroglitazar, BMS986036, ranifibranor, semaglutide, nita GRI_0621, EYP001, VK2809, Daymepen, LIK066, MT_3995, Elovisivats, Namodenosone, Poralu Mab, SAR425899, Sotaglifloin 6865571 NV 556, AZD2693, SP_1373, VK0214, HepaStem, TGFTX4, RLBN1127, GKT_137831, RYI_018, CB4209-CB4211, and JH_0920.

In some embodiments, the therapeutic agent treating the metabolic disorder is a melanocortin 4 receptor (MC4R) agonist. In some embodiments, MC4R agonists include proteins, peptides, nucleic acid molecules or small molecules. In some embodiments, the protein is a peptide analog of MC4R. In some embodiments, the peptide is setmelanotide. In some embodiments, the therapeutic agent for treating or inhibiting type 2 diabetes and/or reducing BMI is setmelanotide and sibutramine, orlistat, phentermine, lorcaserin, naltrexone, liraglutide, diethylpropion, bupropion, metformin , a combination of one or more of pramlintide, topiramate, and zonisamide. In some embodiments, the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. In some embodiments, the small molecule is 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid. In some embodiments, the MC4R agonist is ALB-127158(a).

Examples of therapeutic agents that treat or inhibit cardiomyopathy include: 1) antihypertensive agents such as ACE inhibitors, angiotensin II receptor blockers, beta blockers and calcium channel blockers; 2) agents that slow the heart rate, such as beta blockers, calcium channel blockers and digoxin; 3) agents that keep the heart beating in a normal rhythm, such as antiarrhythmic agents; 4) electrolyte balancing agents such as aldosterone blockers; 5) Agents that remove excess fluid and sodium from the body, such as diuretics; 6) agents that prevent clot formation, such as anticoagulants or blood thinners; and 7) agents that reduce inflammation such as corticosteroids.

Examples of therapeutic agents that treat or inhibit heart failure include, but are not limited to, ACE inhibitors, angiotensin-2 receptor blockers, beta blockers, mineralocorticoid receptor antagonists, diuretics, ivabradine, sacubitril valsartan, hydralazine with nitrates, and digoxin. don't

Examples of therapeutic agents that treat or inhibit hypertension include diuretics (e.g., chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, and metolazone), beta-blockers (e.g., acebutolol, ate nolol, betaxolol, bisoprolol fumarate, carteolol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, etc.), ACE inhibitors (e.g., benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quinapril hydrochloride, ramipril and trandolapril), angiotensin II receptor blockers (eg candesartan, eprosartan mesylate, irve Sartan, losartan potassium, telmisartan, and valsartan), calcium channel blockers (e.g., amlodipine besilate, bepridil, diltiazem hydrochloride, felodipine, isradipine, nicardipine, nifedipine, nisoldipine and verapamil hydrochloride), alpha blockers (eg doxazosin mesylate, prazosin hydrochloride and terazosin hydrochloride), alpha-2 receptor agonists (eg methyldopa), alpha and beta complex blockers (eg carve dilol and labetalol hydrochloride), central agents (e.g., alpha methyldopa, clonidine hydrochloride, guanabenz acetate, and guanfacine hydrochloride), peripheral adrenergic depressants (e.g., guanadrel, guanethidine monosulfate, and serpins) and vasodilators (eg, hydralazine hydrochloride and minoxidil).

In some embodiments, a dose of a therapeutic agent that treats or inhibits a metabolic disorder and/or cardiovascular disease is administered in a subject heterozygous for an INHBE predicted loss-of-function variant compared to a subject who is INHBE baseline (a person who can receive a standard dose). may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% (i.e. lower than the standard dose) . In some embodiments, the dosage of a therapeutic agent that treats or inhibits a metabolic disorder and/or cardiovascular disease can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, subjects heterozygous for INHBE predicted loss-of-function variants may be administered less frequently than subjects with INHBE criteria.

In some embodiments, the dose of a therapeutic agent that treats a metabolic disorder and/or cardiovascular disease is homozygous for a predicted loss-of-function variant INHBE nucleic acid molecule compared to a subject heterozygous for the predicted loss-of-function variant INHBE nucleic acid molecule. About 10%, about 20%, about 30%, about 40%, about 50% for a human subject. In some embodiments, the dosage of a therapeutic agent that treats or inhibits a metabolic disorder and/or cardiovascular disease can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent that treats or inhibits a metabolic disorder and/or cardiovascular disease in a subject homozygous for the predicted loss-of-function variant INHBE nucleic acid molecule can be administered in a subject heterozygous for the predicted loss-of-function variant INHBE nucleic acid molecule. may be administered less frequently than

Administration of a therapeutic agent and/or an INHBE inhibitor for treating or inhibiting metabolic disorders and/or cardiovascular disease may be, for example, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 It can be repeated after weeks, 6 weeks, 7 weeks, 8 weeks, 2 months or 3 months. Repeated administrations can be of the same dose or different doses. Administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. For example, depending on a particular dosing regimen, a subject may receive treatment for an extended period of time, such as for example 6 months, 1 year or more.

Administration of therapeutic agents and/or INHBE inhibitors for treating or inhibiting metabolic disorders and/or cardiovascular disease may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. It can occur by any suitable pathway, including but not limited to. Pharmaceutical compositions for administration are preferably sterile, substantially isotonic, and prepared under GMP conditions. A pharmaceutical composition may be presented in unit dosage form (ie, a dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or adjuvants. Formulations depend on the route of administration chosen. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and is not substantially injurious to the recipient.

As used herein, the terms "treat", "treating" and "treatment" and "prevent", "preventing" and "prophylaxis" refer to inducing a desired biological response, such as a therapeutic and prophylactic effect, respectively. In some embodiments, the therapeutic effect is a reduction/reduction in metabolic disorder and/or cardiovascular disease, a reduction/decrease in severity of metabolic disorder and/or cardiovascular disease (e.g., a metabolic disorder and/or reduction or inhibition of development of cardiovascular disease), reduction/reduction of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects, reduction of onset of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects. /reduction, delay in onset of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects, reduction in severity of symptoms of metabolic-disorder related effects and/or cardiovascular disease-related effects, symptoms and metabolic disorder-related effects, and/or or reducing the number of cardiovascular disease-related effects, reducing the latency of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects, alleviating symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects, reducing secondary symptoms, secondary reducing infection, preventing recurrence to metabolic disorders and/or cardiovascular disease, reducing the number or frequency of recurrent episodes, increasing the incubation period between symptom episodes, increasing the time to sustained progression, accelerating recovery, or increasing the efficacy or reducing tolerance of alternative therapies; and/or increased survival time of infected host animals. A prophylactic effect may include complete or partial avoidance/inhibition or delay (e.g., complete or partial avoidance/inhibition or delay) of metabolic disorders and/or cardiovascular disease onset/progression after administration of the treatment protocol and an increase in affected host animals. survival time may be included. Treatment of a metabolic disorder is the treatment, initiation or progression of a subject already diagnosed as having any form of metabolic disorder and/or cardiovascular disease at any clinical stage or indication or worsening of symptoms or signs of a metabolic disorder and/or cardiovascular disease. or delaying the decline, and/or preventing and/or reducing the severity of metabolic disorders and/or cardiovascular disease.

The present disclosure also provides methods for identifying a subject at increased risk of developing a metabolic disorder. In some embodiments, a method determines the presence or absence of an INHBE variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and/or cDNA molecule) encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject; including what has been decided If a subject lacks an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide (ie, an individual is genotyped on the basis of INHBE), the subject is at increased risk of developing a metabolic disorder. When the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide (i.e., the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide), the subject reduces the risk of developing metabolic disorders. In some embodiments, liver expression quantitative trait loci (eQTLs) can be analyzed.

The present disclosure also provides methods for identifying a subject at increased risk of developing a cardiovascular disease. In some embodiments, a method determines the presence or absence of an INHBE variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and/or cDNA molecule) encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject; including what has been decided If a subject lacks an INHBE variant nucleic acid molecule that encodes an INHBE predicted loss-of-function polypeptide (ie, if an individual is genotyped on the basis of INHBE), the subject is at increased risk of developing cardiovascular disease. When the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide (i.e., the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide), the subject reduces the risk of developing cardiovascular disease. In some embodiments, liver expression quantitative trait loci (eQTLs) can be analyzed.

Having a single copy of the INHBE variant nucleic acid molecule encoding the INHBE predicted loss-of-function polypeptide results in metabolic disorders and/or cardiovascular disease than not having a copy of the INHBE variant nucleic acid molecule encoding the INHBE predicted loss-of-function polypeptide. further protects the subject from Without being limited to any particular theory or mechanism of action, it is believed that a single copy of the INHBE variant nucleic acid molecule (i.e., heterozygous for the INHBE variant nucleic acid molecule) protects a subject from developing metabolic disorders and/or cardiovascular disease, and also It is believed that having two copies of the INHBE variant nucleic acid molecule (i.e., homozygous for the INHBE variant nucleic acid molecule) protects a subject more from developing metabolic disorders and/or cardiovascular disease compared to a subject with a single copy. Thus, in some embodiments, a single copy of an INHBE variant nucleic acid molecule may not completely protect, but may partially or incompletely protect a subject from developing a metabolic disorder and/or cardiovascular disease. Without wishing to be bound by any particular theory, there may be additional factors or molecules associated with the development of metabolic disorders and/or cardiovascular disease that are still present in subjects having a single copy of the INHBE variant nucleic acid molecule, and thus resulting in metabolic disorders and/or not completely protected from developing cardiovascular disease.

determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample from the subject and/or whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide Determining may be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these examples, the nucleic acid molecule can be present in a cell obtained from a subject.

In some embodiments, if the subject is determined to be at increased risk of developing a metabolic disorder, the subject is further treated with a therapeutic agent that treats or inhibits the metabolic disorder and/or an INHBE inhibitor as described herein. For example, if a subject is on INHBE criteria and is therefore at increased risk of developing a metabolic disorder, the subject is administered an INHBE inhibitor. In some embodiments, such subjects are also administered a therapeutic agent that treats or inhibits a metabolic disorder. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits a metabolic disorder at a dose equal to or less than a standard dose. and an INHBE inhibitor is administered. In some embodiments, such subjects are also administered a therapeutic agent that treats or inhibits a metabolic disorder. In some embodiments, where the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits a metabolic disorder at a dose equal to or less than the standard dose. receive In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. In some embodiments, the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In some embodiments, if the subject is determined to be at increased risk of developing cardiovascular disease, the subject is further treated with a therapeutic agent that treats or inhibits cardiovascular disease and/or an INHBE inhibitor as described herein. For example, if the subject is on INHBE criteria and therefore has an increased risk of developing cardiovascular disease, the subject is administered an INHBE inhibitor. In some embodiments, such subjects are also administered a therapeutic agent that treats or inhibits cardiovascular disease. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits cardiovascular disease at a dose equal to or less than a standard dose. and an INHBE inhibitor is administered. In some embodiments, such subjects are also administered a therapeutic agent that treats or inhibits cardiovascular disease. In some embodiments, where the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits cardiovascular disease at a dose equal to or less than the standard dose. receive In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. In some embodiments, the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In some embodiments, any of the methods described herein can be used to generate an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide and/or an INHBE predicted loss-of-function variant polypeptide associated with a reduced risk of developing a metabolic disorder and/or cardiovascular disease. It may further include determining the genetic burden of a subject having. Gene burden is the collection of all variants of the INHBE gene that can be performed in an association analysis with metabolic disorders and/or cardiovascular disease. In some embodiments, the subject is homozygous for one or more INHBE variant nucleic acid molecules encoding an INHBE predicted loss-of-function polypeptide associated with a metabolic disorder and/or reduced risk of developing cardiovascular disease. In some embodiments, the subject is heterozygous for one or more INHBE variant nucleic acid molecules encoding an INHBE predicted loss-of-function polypeptide associated with a metabolic disorder and/or reduced risk of developing cardiovascular disease. The results of association analysis suggest that INHBE variant nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides are associated with reduced risk of developing metabolic disorders and/or cardiovascular disease. When a subject's genetic burden is low, the subject has a higher risk of developing a metabolic disorder and/or cardiovascular disease, and the subject is treated with a standard dose of a therapeutic agent and/or INHBE that treats, prevents or inhibits the metabolic disorder and/or cardiovascular disease. Receive or continue to receive inhibitors. When a subject has a greater genetic burden, the subject has a lower risk of developing a metabolic disorder and/or cardiovascular disease, and the subject is treated, prevented, or inhibited a metabolic disorder and/or cardiovascular disease in a sub-standard dosage amount. Treatment is administered or continues to be administered. The greater the genetic burden, the lower the risk of developing metabolic disorders and/or cardiovascular disease.

In some embodiments, the genetic burden of a subject having any one or more INHBE variant nucleic acid molecules encoding an INHBE predicted loss-of-function polypeptide is a weighted majority of any INHBE variant nucleic acid molecules encoding an INHBE predicted loss-of-function polypeptide. represents the sum. In some embodiments, the genetic burden is at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 2, at least about 3, at least about 4, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, At least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more genetic variants and genetic burden calculated using an estimate of association with a metabolic disorder or related outcome (e.g., a weighted burden score) for each allele is the number of alleles multiplied by This may include all genetic variants, regardless of genomic annotation, that are close to the INHBE gene (up to 10 Mb around the gene) that show non-zero association with metabolic disorder-related traits and/or cardiovascular disease-related traits in genetic association analysis. . In some embodiments, if the subject has a genetic burden equal to or greater than a desired threshold score, the subject has a reduced risk of developing a metabolic disorder and/or cardiovascular disease. In some embodiments, if the subject has a genetic burden below a desired threshold score, the subject has an increased risk of developing a metabolic disorder and/or cardiovascular disease.

In some embodiments, genetic burden can be divided into quintiles, eg, upper quintile, middle quintile, and lower quintile, wherein the upper quintile of genetic burden corresponds to the lowest risk group and the lower quintile of genetic burden The fifth quartile corresponds to the high-risk group. In some embodiments, a subject with a greater genetic burden has the highest weighted genetic burden, including but not limited to, the top 10%, the top 20%, the top 30%, the top 40%, or the top 50% of the genetic burden in a population of subjects. includes In some embodiments, the genetic variation is a genetic variation associated with a metabolic disorder and/or cardiovascular disease in the top 10%, top 20%, top 30%, top 40%, or top 50% of a p-value range for association. includes In some embodiments, each identified genetic variation has a p-value of about 10 ⁻² , about 10 ⁻³ , about 10 ⁻⁴ , about 10 ⁻⁵ , about 10 ⁻⁶ , about 10 ⁻⁷ , about 10 ^{− 8} , about 10 ^-9 , about 10 ^-10 , about 10 ^-11 , about 10 ^-12 , about 10 ^-13 , about 10 ^-14 , or about 10 ^-15 genetic variants associated with metabolic disorders and/or cardiovascular disease includes In some embodiments, the genetic variation identified comprises a genetic variation associated with a metabolic disorder and/or cardiovascular disease with a p-value of less than 5 x 10 ⁻⁸ . In some embodiments, the identified genetic variance is about 1.5 or greater, about 1.75, about 2.0 or greater, or about 2.25 or greater for the top 20% of the distribution compared to the rest of the reference population; or a genetic variant associated with a metabolic disorder and/or cardiovascular disease in a high-risk subject with an odds ratio (OR) of about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater. In some embodiments, the odds ratio (OR) is about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or greater than 7.0. In some embodiments, high-risk subjects include subjects with a genetic burden in the bottom decile, quintile, or tertiary of a reference population. Thresholds for gene burden are determined based on the nature of the intended practical application and the difference in risk considered significant for that practical application.

In some embodiments, if the subject is identified as having an increased risk of developing a metabolic disorder, the subject is further treated with a therapeutic agent and/or an INHBE inhibitor that treats, prevents or inhibits the metabolic disorder as described herein. For example, an INHBE inhibitor is administered to a subject if the subject is on INHBE criteria and therefore has an increased risk of developing a metabolic disorder. In some embodiments, such subjects are also administered a therapeutic agent that treats or inhibits a metabolic disorder. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits a metabolic disorder at a dose equal to or less than the standard dose. and an INHBE inhibitor is administered. In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. In addition, if a subject has a lower genetic burden with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, and thus is at increased risk of developing a metabolic disorder, the subject is administered a therapeutic agent that treats, prevents or inhibits the metabolic disorder. receive In some embodiments, if the subject has a lower genetic burden with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject has a larger INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. Subjects with a genetic burden are administered a therapeutic agent that treats, prevents or inhibits a metabolic disorder at a dose equal to or greater than a standard dose.

In some embodiments, if the subject is identified as having an increased risk of developing cardiovascular disease, the subject is further treated with a therapeutic agent that treats, prevents or inhibits cardiovascular disease and/or an INHBE inhibitor as described herein. For example, an INHBE inhibitor is administered to a subject if the subject is on INHBE criteria and therefore has an increased risk of developing a cardiovascular disease. In some embodiments, such subjects are also administered a therapeutic agent that treats, prevents or inhibits cardiovascular disease. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats, prevents or inhibits cardiovascular disease at a dose equal to or less than a standard dose. , and an INHBE inhibitor is also administered. In some embodiments, the subject is on INHBE criteria. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. In addition, if a subject has a lower genetic burden with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, and thus is at increased risk of developing cardiovascular disease, the subject may seek a therapeutic agent that treats, prevents or inhibits cardiovascular disease. get dosed In some embodiments, if the subject has a lower genetic burden with an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject has a larger INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. Subjects with a genetic burden are administered a therapeutic agent that treats, prevents or inhibits cardiovascular disease at a dose equal to or greater than a standard dose.

The present disclosure also provides methods for diagnosing a metabolic disorder in a subject. Methods include determining or determining whether a subject has any one or more of the INHBE variant nucleic acid molecules described herein or polypeptides generated therefrom. If a subject is on INHBE criteria and has one or more symptoms of a metabolic disorder, the subject is diagnosed as having a metabolic disorder. In some embodiments, the subject is homozygous for a reference INHBE nucleic acid molecule. In some embodiments, the subject is homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide. In some embodiments, if the subject is identified as having a metabolic disorder (such as having one or more symptoms of a metabolic disorder and being homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide), The subject is further treated with a therapeutic agent that treats or inhibits a metabolic disorder, such as any of those described herein.

The present disclosure also provides methods for diagnosing cardiovascular disease in a subject. Methods include determining or determining whether a subject has any one or more of the INHBE variant nucleic acid molecules described herein or polypeptides generated therefrom. If a subject is on INHBE criteria and has one or more symptoms of cardiovascular disease, the subject is diagnosed as having cardiovascular disease. In some embodiments, the subject is homozygous for a reference INHBE nucleic acid molecule. In some embodiments, the subject is homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide. In some embodiments, if the subject is identified as having cardiovascular disease (such as having one or more symptoms of cardiovascular disease and being homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide), The subject is further treated with a therapeutic agent that treats or inhibits a cardiovascular disease, such as any described herein.

The disclosure also provides a method for identifying a subject at increased risk of developing a metabolic disorder, wherein the method comprises determining or determining the presence or absence of an INHBE predicted loss-of-function polypeptide in a biological sample obtained from the subject. . In some embodiments, the method is a blood-based quantitative assay, such as a somatic cell assay to quantify Inhibin E.

The present disclosure also provides a method for identifying a subject at increased risk of developing a cardiovascular disorder, wherein the method comprises determining or determining the presence or absence of an INHBE predicted loss-of-function polypeptide in a biological sample obtained from the subject. . In some embodiments, the method is a blood-based quantitative assay, such as a somatic cell assay to quantify Inhibin E.

The presence of an INHBE polypeptide in an appropriate fluid sample such as blood, plasma and/or serum can be determined by detecting the INHBE polypeptide using a variety of methods for measuring INHBE or INHBE activity. For example, INHBE polypeptides can be detected in an immunoassay using an antibody specific for INHBE. Antibodies can selectively bind to INHBE polypeptides and/or CEA. Antibodies can be used, for example, in Western blots of one-dimensional or two-dimensional gels, high-throughput methods such as enzyme-linked immunoassays, and/or dot blot (antibody sandwich) assays of whole cell proteins or partially purified proteins. In some embodiments, the concentration of INHBE in a suitable fluid is measured by enzyme-linked immunosorbent assay (ELISA). In one example of the assay, a serum sample is diluted 400-fold and applied to a plate with an INHBE polypeptide antibody (primary antibody) from one animal. INHBE can bind to these INHBE antibodies if sufficient INHBE is present in the serum. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody", such as an antibody of animal origin different from the primary antibody, an antibody that binds to the primary antibody, is applied to the plate and then washed again. This secondary antibody is previously chemically linked to, for example, an enzyme. Thus, the plate will contain enzymes in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and the color or fluorescence changes due to the catalyzed action by the enzyme. A sample that produces a stronger signal than a known healthy sample is "positive". Anything that produces a weaker signal than a known healthy sample is "negative".

Alternatively, the concentration of the INHBE polypeptide in a suitable fluid may be determined using a spectroscopic method such as the SDS PAGE method or a similar method to quantify proteins, which is later quantified by LC-MS/MS mass spectrometry, GCMS mass spectrometry, densitometric or mass spectrometry methods. It can be determined by detecting the INHBE polypeptide. Additional methods for quantifying polypeptide levels include HPLC (high performance liquid chromatography), SEC (size exclusion chromatography), modified Lowry assay, spectrophotometry, SEC-MALLS (size exclusion chromatography/multiangle laser light scattering), and Nuclear Magnetic Resonance (NMR) is included, but is not limited thereto.

Aptamers specific for INHBE polypeptides may also be used. A suitable aptamer can selectively bind an INHBE polypeptide to measure blood, plasma or serum concentrations of the INHBE polypeptide or to detect the presence of variant INHBE. INHBE polypeptides produced recombinantly or by chemical synthesis, fragments thereof, including fusion proteins, or other derivatives or analogs thereof can be used as immunogens to generate aptamers that recognize the INHBE polypeptides. The term “aptamer” refers to a non-naturally occurring oligonucleotide chain or peptide molecule that has a specific action for a target compound (eg, a specific epitope, therapeutic drug marker or surrogate marker). Specific action includes, but is not limited to, binding of the target compound, catalytically changing the target compound, and/or reacting with the target compound in a manner that modifies/alteres the target compound or a functional activity of the target compound. Aptamers can be engineered through iterative rounds of in vitro selection or SELEX ^™ (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, such as small molecules. Production/synthesis methods are described in the literature (Ellington et al., Nature, 1990, 346, 818-822; and Tuerk et al., Science, 1990, 249, 505-510). The "SELEX ^™ " methodology involves the amplification of the selected nucleic acid with the combination of selected nucleic acid ligands that interact with a specific epitope in a desired action, eg, binding to a protein. Any iterative cycle of the selection/amplification step allows selection of one or a few nucleic acids that interact most strongly with a particular epitope from a pool containing a very large number of nucleic acids. The cycle of selection/amplification procedures continues until the selected goal is achieved. The SELEX methodology is covered by US Patent US Pat. 5,475,096 and 5,270,163.

The present disclosure also provides methods for identifying a subject having a disease, such as a metabolic disorder, that may respond differentially to treatment with an INHBE inhibitor or other therapeutic agent that affects fat distribution. In some embodiments, a method involves the presence or absence of INHBE pLOF or pGOF in a biological sample (liver, plasma, serum and/or whole blood) obtained from a subject or INHBE pLOF associated with hepatic expression of INHBE or measurement of INHBE in the circulation or expression in the liver. or having determined or determined the presence or absence of pGOF. If a subject lacks this INHBE variant (i.e., the subject is genotypically classified based on INHBE), the subject has an increased risk of developing a metabolic disorder and may be treated with an INHBE inhibitor or other treatment that affects fat distribution. When a subject has such an INHBE variant nucleic acid molecule (ie, when the subject is heterozygous for INHBE pLOF/pGOF or homozygous for INHBE pLOF/pGOF), the subject is at reduced risk of developing a metabolic disorder.

The present disclosure also provides methods of detecting the presence or absence of an INHBE variant nucleic acid molecule (genomic, mRNA or cDNA) encoding a predicted loss-of-function INHBE polypeptide in a biological sample from a subject. It is understood that the sequences of genes within a population and the mRNA molecules encoded by those genes may change due to polymorphisms, such as single-nucleotide polymorphisms.

A biological sample can be derived from any cell, tissue or biological fluid from a subject. The sample may include any clinically relevant tissue such as a bone marrow sample, tumor biopsy, fine needle aspirate, or bodily fluid sample such as blood, gingival sulcus fluid, plasma, serum, lymph fluid, ascites fluid, cystic fluid or urine. there is. In some cases, the sample consists of a buccal swab. The sample used in the methods disclosed herein will vary depending on the assay format, the nature of the detection method, and the tissue, cell or extract used as the sample. Biological samples may be treated differently depending on the assay used. For example, when detecting any predicted loss-of-function variant INHBE nucleic acid molecule, a preprocess designed to isolate or enrich the sample for genomic DNA can be used. Various techniques can be used for this. When detecting levels of the predicted loss-of-function variant INHBE mRNA, a variety of techniques can be used to enrich biological samples with mRNA. A variety of methods can be used to detect the presence or level of mRNA or the presence of a particular variant genomic DNA locus.

In some embodiments, detecting an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide in a subject comprises an INHBE genomic nucleic acid molecule in a biological sample and/or an INHBE mRNA molecule in a biological sample, and/or an mRNA molecule in a biological sample. comprising assaying or genotyping a biological sample obtained from a subject to determine whether an INHBE cDNA molecule generated from a subject causes loss-of-function (partial or complete) or a predicted loss-of-function INHBE described herein As with any INHBE variant nucleic acid molecule encoding a polypeptide, it contains one or more mutations predicted to result in loss-of-function (partial or complete).

In some embodiments, a method for detecting the presence or absence of INHBE variant nucleic acid molecules (eg, genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules generated from mRNA molecules) in a subject is a biological sample obtained from the subject. It includes performing a test for The assay determines whether a nucleic acid molecule in a biological sample contains a particular nucleotide sequence.

In some embodiments, a biological sample comprises cells or cell lysates. Such a method may include, for example, obtaining a biological sample from a subject, including INHBE genomic nucleic acid molecules or mRNA molecules and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such an assay may include, for example, determining the identity of this position of a particular INHBE nucleic acid molecule. In some embodiments, the method is an in vitro method.

In some embodiments, the determining step, detecting step, or genotyping comprises sequencing at least a portion of a nucleotide sequence of an INHBE genomic nucleic acid molecule, an INHBE mRNA molecule, or an INHBE cDNA molecule in a biological sample, wherein the sequenced portion is a loss-of-function (partial or complete) or predicted to cause loss-of-function (partial or complete), e.g., predicted loss-of-function variants INHBE nucleic acid molecules described herein.

In some embodiments, the determining step, detection step or genotyping assay is performed by determining the nucleotide sequence of an INHBE genomic nucleic acid molecule in a biological sample, the nucleotide sequence of an INHBE mRNA molecule in a biological sample, or the nucleotide sequence of an INHBE cDNA molecule generated from INHBE mRNA in a biological sample. Sequencing at least a portion of In some embodiments, the determining step, detection step or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule in a biological sample. In some embodiments, the determining step, detection step or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample. In some embodiments, the determining step, detection step or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an INHBE cDNA molecule generated from an INHBE mRNA molecule in a biological sample.

In some embodiments, an assay comprises sequencing entire nucleic acid molecules. In some embodiments, only INHBE genomic nucleic acid molecules are analyzed. In some embodiments, only INHBE mRNA is analyzed. In some embodiments, only INHBE cDNA obtained from INHBE mRNA is analyzed.

In some embodiments, the determining step, detection step or genotyping assay comprises: a) amplifying at least a portion of a nucleic acid molecule encoding an INHBE polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and d) detecting the detectable label.

In some embodiments, the nucleic acid molecule is an mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplification step.

In some embodiments, the determining step, detection step, or genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with an alteration-specific probe comprising a detectable label, wherein the alteration-specific probe amplifies under stringent conditions. a nucleotide sequence that hybridizes to a nucleotide sequence of a nucleic acid molecule; and detecting the detectable label. Strain-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleic acid sequences. Strain-specific primers can be used because DNA polymerase does not expand in the presence of a mismatch with the template.

In some embodiments, the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is in a cell obtained from a subject.

In some embodiments, the assay is a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes under stringent conditions to an INHBE variant nucleic acid molecule (genomic, mRNA or cDNA) but not to the corresponding INHBE reference sequence. contacting the biological sample with the step of determining whether hybridization has occurred. In some embodiments, an assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also includes reverse transcription of mRNA into cDNA, eg, by reverse transcriptase polymerase chain reaction (RT-PCR).

In some embodiments, the method is capable of specifically detecting and/or identifying a polynucleotide comprising an INHBE variant nucleic acid molecule (genomic, mRNA or cDNA) that binds a target nucleotide sequence and encodes a predicted loss-of-function INHBE polypeptide. Probes and primers of sufficient nucleotide length are used. Hybridization conditions or reaction conditions can be determined by the operator to achieve these results. The nucleotide length may be any length sufficient for use in the selected detection method, including any assay described or exemplified herein. These probes and primers are capable of specifically hybridizing to target nucleotide sequences under high stringency hybridization conditions. Although probes that are different from the target nucleotide sequence and retain the ability to specifically detect and/or identify the target nucleotide sequence can be designed by conventional methods, probes and primers are capable of maintaining complete nucleotide sequence identity of contiguous nucleotides within the target nucleotide sequence. can have Probes and primers are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% of the nucleotide sequence of the target nucleic acid molecule. , about 98%, about 99% or 100% sequence identity or complementarity.

Illustrative examples of nucleic acid sequencing technologies include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing, which use labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). It includes doing In some methods, a target nucleic acid molecule can be amplified prior to or concurrently with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

In hybridization techniques, stringent conditions may be used to allow a probe or primer to specifically hybridize to its target. In some embodiments, polynucleotide primers or probes under stringent conditions are detectably greater than other non-target sequences, e.g., by at least 2-fold, at least 3-fold, at least 4-fold or more, including 10-fold or more, background. will hybridize to its target sequence to a greater degree than background. In some embodiments, under stringent conditions a polynucleotide primer or probe will hybridize to its target nucleotide sequence to a detectably greater extent than another nucleotide sequence by at least twice as much. In some embodiments, under stringent conditions a polynucleotide primer or probe will hybridize to its target nucleotide sequence to a detectably greater extent than another nucleotide sequence by at least 3-fold. In some embodiments, under stringent conditions a polynucleotide primer or probe will hybridize to its target nucleotide sequence to a detectably greater extent than another nucleotide sequence by at least 4-fold. In some embodiments, under stringent conditions a polynucleotide primer or probe will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences by a factor of 10 above background. Stringent conditions are sequence dependent and situation dependent.

Appropriate stringency conditions that promote DNA hybridization are known or can be found in the literature, e.g., 6X sodium chloride/sodium citrate (SSC) at about 45°C followed by a wash of 2X SSC at 50°C ( Current Protocols in Molecular Biology , John Wiley & Sons, NY (1989), 6.3.1-6.3.6). Typically, stringent conditions for hybridization and detection are a salt concentration of less than about 1.5 M Na ⁺ ions, typically about 0.01 to 1.0 M Na ⁺ ions (or other salts) at pH 7.0 to 8.3, and a short probe (e.g., , 10 to 50 nucleotides), the temperature is at least about 30°C, and for longer probes (eg, greater than 50 nucleotides) at least about 60°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may include about 0.1% to about 1% SDS. The hybridization period is generally less than about 24 hours, usually about 4 to about 12 hours. Washing time should be at least sufficient to reach equilibrium.

The present disclosure also provides a sample obtained from a subject to determine whether an INHBE polypeptide in the subject contains one or more variances that result in a polypeptide having loss-of-function (partial or complete) or predicted loss-of-function (partial or complete). Provided is a method of detecting the presence of a human INHBE predicted loss-of-function polypeptide comprising performing an assay for.

In some embodiments, detecting comprises sequencing at least a portion of the polypeptide. In some embodiments, the detecting step comprises an immunoassay to detect the presence of the polypeptide.

In some embodiments, when the subject does not have an INHBE predicted loss-of-function polypeptide, the subject has a metabolic disorder or any type 2 diabetes, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as , ALT and/or AST), obesity, hypertension, NASH and/or elevated triglyceride levels. In some embodiments, when the subject has an INHBE predicted loss-of-function polypeptide, the subject has a metabolic disorder or any type 2 diabetes mellitus, obesity, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes ( eg ALT and/or AST), high blood pressure, NASH and/or elevated triglyceride levels.

In some embodiments, if the subject does not have an INHBE predicted loss-of-function polypeptide, the subject has an increased risk of developing a cardiovascular disease or any cardiomyopathy, heart failure, and hypertension. In some embodiments, when the subject has an INHBE predicted loss-of-function polypeptide, the risk of the subject developing cardiovascular disease or any cardiomyopathy, heart failure, and hypertension is reduced.

The present disclosure also provides an isolation that hybridizes to an INHBE variant genomic nucleic acid molecule, an INHBE variant mRNA molecule, and/or an INHBE variant cDNA molecule (eg, any genomic variant nucleic acid molecule, mRNA variant molecule, and cDNA variant molecule disclosed herein). Use of the nucleic acid molecule described herein is provided in any of the methods described herein.

In some embodiments, such isolated nucleic acid molecules are at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 , at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 , at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75 , at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600 , at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules are at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 , at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 comprises or consists of two nucleotides. In some embodiments, an isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, an isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecules are about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, About 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about It comprises or consists of 18 to about 22 nucleotides. In some embodiments, an isolated nucleic acid molecule consists of or comprises about 18 to about 30 nucleotides. In some embodiments, an isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

In some embodiments, such isolated nucleic acid molecules hybridize to INHBE variant nucleic acid molecules (eg, genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules) under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, alter-specific probes or alter-specific primers described or exemplified herein, including without limitation primers, probes, antisense RNA, shRNA and siRNA; Each is described in more detail elsewhere herein and can be used in any of the methods described herein.

In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about an INHBE variant genomic nucleic acid molecule, an INHBE variant mRNA molecule, and/or an INHBE variant cDNA molecule. It hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical. In some embodiments, an isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, an isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, an isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

In some embodiments, an alteration-specific probe and an alteration-specific primer comprise DNA. In some embodiments, an alteration-specific probe and an alteration-specific primer comprise RNA.

In some embodiments, probes and primers described herein (including alteration-specific probes and alteration-specific primers) have nucleotide sequences that specifically hybridize to any nucleic acid molecule or its complement disclosed herein. In some embodiments, probes and primers specifically hybridize under stringent conditions to any nucleic acid molecule disclosed herein.

In some embodiments, primers comprising alteration-specific primers can be used for second generation sequencing or high throughput sequencing. In some cases, primers, including modification-specific primers, may be modified. In particular, the primers may include various modifications used in different steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing and 454 Pyrosequencing. Modified primers can be used at different stages of the process, including biotinylated primers in the cloning step and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is usually performed using paired-end tagged libraries where each molecule of the DNA template is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate oligonucleotides are used in the detection step. The adapter may contain a 5'-biotin tag for immobilizing the DNA library on streptavidin-coated beads.

The probes and primers described herein can be used to detect nucleotide variations within any of the INHBE variant genomic nucleic acid molecules, INHBE variant mRNA molecules, and/or INHBE variant cDNA molecules disclosed herein. The primers described herein can be used to amplify INHBE variant genomic nucleic acid molecules, INHBE variant mRNA molecules, or INHBE variant cDNA molecules or fragments thereof.

A probe or primer (e.g., an alteration-specific probe or an alteration-specific primer) in the context of the present disclosure refers to an INHBE reference genomic nucleic acid molecule, an INHBE reference mRNA molecule, and/or an INHBE It means that it does not hybridize to the nucleic acid sequence encoding the reference cDNA molecule.

In some embodiments, a probe (eg, an alteration-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, radiolabel or biotin.

The present disclosure also provides a support comprising a substrate to which any one or more probes disclosed herein are attached. A solid support is a solid-state substrate or support to which molecules such as any of the probes disclosed herein can be associated. One type of solid support is an array. Another type of solid support is an array detector. An array detector is a solid support to which several different probes are coupled into an array, grid, or other organized pattern. One type of solid-state substrate is a microtiter dish, such as the standard 96-well type. In some implementations, multiwell glass slides may be used, generally containing one array per well.

The nucleotide sequence of the INHBE reference genome nucleic acid molecule is set forth in SEQ ID NO: 1 (ENST00000266646.3 including chr12:57455307-57458025 in the GRCh38/hg38 Human Genome Assembly).

The nucleotide sequence of the INHBE reference mRNA molecule is shown in SEQ ID NO:2. The nucleotide sequence of another INHBE reference mRNA molecule is set forth in SEQ ID NO:3. The nucleotide sequence of another INHBE reference mRNA molecule is set forth in SEQ ID NO:4.

The nucleotide sequence of the INHBE reference cDNA molecule is set forth in SEQ ID NO:5. The nucleotide sequence of another INHBE reference cDNA molecule is set forth in SEQ ID NO:6. The nucleotide sequence of another INHBE reference cDNA molecule is set forth in SEQ ID NO:7.

The amino acid sequence of the INHBE reference polypeptide is set forth in SEQ ID NO:8. Referring to SEQ ID NO: 8, the INHBE reference polypeptide is 350 amino acids in length.

Genomic nucleic acid molecules, mRNA molecules and cDNA molecules can be from any organism. For example, genomic nucleic acid molecules, mRNA molecules and cDNA molecules can be orthologs from other organisms such as humans or non-human mammals, rodents, mice or rats. It is understood that genetic sequences within a population may change due to polymorphisms, such as single-nucleotide polymorphisms. The examples provided herein are merely illustrative sequences. Other sequences are also possible.

An isolated nucleic acid molecule disclosed herein may include RNA, DNA, or both RNA and DNA. An isolated nucleic acid molecule may be linked or fused to a heterologous nucleic acid sequence, such as a vector or heterologous marker. For example, an isolated nucleic acid molecule disclosed herein may exist within a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. An isolated nucleic acid molecule may also be linked or fused to a heterologous label. A label may be directly detectable (eg, a fluorophore) or indirectly detectable (eg, a hapten, enzyme, or fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels and fluorescent labels. Labels include, for example, chemiluminescent materials; metal-containing materials; or an enzyme in which the enzyme-dependent secondary production of the signal occurs. The term "label" can also refer to a "tag" or hapten capable of selectively binding to a conjugated molecule such that it is used to generate a detectable signal when the conjugated molecule is subsequently added with a substrate. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish hydrogen peroxide (HRP) to bind to the tag, and a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or HRP's It can be tested using a fluorescent substrate to detect its presence. Exemplary labels that can be used as tags to facilitate purification include myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags or Fc of immunoglobulins. Including but not limited to parts. A number of labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

The disclosed nucleic acid molecules may include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutions. Such nucleotides include nucleotides that contain modified bases, sugar or phosphate groups or contain unnatural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

A nucleic acid molecule disclosed herein may also contain one or more nucleotide analogs or substitutions. Nucleotide analogues are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to base moieties include, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenine, as well as natural and synthetic modifications of A, C, G and T/U. different purine or pyrimidine bases such as -9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, adenine and guanine. 2-propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-haluracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo (e.g., 5 -bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaguanine but is not limited to azaadenine, 3-deazaguanine and 3-deazaadenine.

Nucleotide analogs may also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C _1-10 alkyl or C _2-10 alkenyl, and C _2-10 alkynyl. Exemplary 2' sugar modifications are also -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n -ONH ₂ , and -O(CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from 1 to about 10. Other modifications at the 2' position include C _1-10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group , reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacokinetic properties of oligonucleotides and other substituents with similar properties. Similar modifications can be made at other positions of the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Modified sugars may also include those containing modifications at the bridging ring oxygens, such as CH ₂ and S. A nucleotide sugar analogue may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

Nucleotide analogues can also be modified at the phosphate moiety. Modified phosphate moieties are those in which the linkage between two nucleotides is phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and 3'-alkylene phosphonate and other alkyl phosphonates, including chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thiono alkylphosphonates, thionoalkylphosphotriesters, and those that may be modified to contain boranophosphates. This phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, where the linkage is 3'-5' to 5'-3' or 2'-5' may contain an inverted polarity, such as from to 5'-2'. Various salts, mixed salts and free acid forms are also included. Nucleotide substitutions also include peptide nucleic acids (PNAs).

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding predicted loss-of-function INHBE polypeptides; INHBE variant mRNA molecules encoding predicted loss-of-function INHBE polypeptides; Or a therapeutic agent that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having an INHBE variant cDNA molecule encoding a predicted loss-of-function INHBE polypeptide.

In some embodiments, the metabolic disorder is type 2 diabetes, and the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, sitagliptin , saxagliptin, linagliptin, exenatide, liraglutide, semaglutide, canagliflozin, dapagliflozin and empagliflozin.

In some embodiments, the metabolic disorder is obesity, and the treatment is selected from orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide.

In some embodiments, the metabolic disorder is hypertension and the treatment is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carte olol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quinapril hydrochloride, ramipril , trandolapril, candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, bepridil, diltiazem hydrochloride, felodipine, isradipine , nicardipine, nifedipine, nisoldipine, verapamil hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol labetalol hydrochloride, alpha methyldopa, clonidine hydrochloride, guanabenz acetate, guanfacine hydrochloride, guanadrel, guanethidine monosulfate and reserpine, hydralazine hydrochloride and minoxidil.

In some embodiments, the metabolic disorder is elevated triglycerides, and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibric acid, niacin, and omega-3 fatty acids.

In some embodiments, the metabolic disorder is lipodystrophy and the therapeutic agent is EGRIFTA ^® (tesamorelin), GLUCOPHAGE ^® (metformin), SCULPTRA ^® (poly-L-lactic acid), RADIESSE ^® (calcium hydroxyapatite), polymethylmetha acrylates (eg PMMA), ZYDERM ^® (bovine collagen), COSMODERM ^® (human collagen), silicones, and hyaluronic acid. In some embodiments, the therapeutic agent that treats or inhibits lipodystrophy includes tesamorelin, metformin, poly-L-lactic acid, calcium hydroxyapatite, polymethylmethacrylate, bovine collagen, human collagen, silicone, and hyaluronic acid. Not limited to this.

In some embodiments, the metabolic disorder is liver inflammation and the therapeutic agent is selected from a hepatitis therapeutic agent and a hepatitis vaccine.

In some embodiments, the metabolic disorder is fatty liver disease and the treatment or procedure is bariatric surgery and/or dietary intervention.

In some embodiments, the metabolic disorder is hypercholesterolemia and the therapeutic agent is a statin (e.g., ^LIPITOR® (atorvastatin), ^LESCOL® (fluvastatin), lovastatin, ^LIVALO® (pitavastatin), ^PRAVACHOL® (pravastatin), ^CRESTOR® (rosuvastatin calcium), and ^ZOCOR® (simvastatin)); bile acid sequestrants (eg, ^PREVALITE® (cholestyramine), ^WELCHOL® (colesevelam) and ^COLESTID® (colestipol)); PCSK9 inhibitors (eg, ^PRALUENT® (alirocumab) and ^REPATHA® (evolocumab)); niacin (eg, niaspan and niacor); fibrates (eg fenofibrate and LOPID ^® (gemfibrozil)) and ATP citrate lyase (ACL) inhibitors (eg NEXLETOL ^® (bempedoic)). In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is a statin (eg, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, and simvastatin); bile acid sequestrants (eg cholestyramine, colesevelam and colestipol); PCSK9 inhibitors (eg, alirocumab and evolocumab); niacin (eg, niaspan and niacor); fibrates (eg, fenofibrate and gemfibrozil); and ACL inhibitors (eg, bempedo acid). In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is alirocumab or evolocumab. In some embodiments, the therapeutic agent treating or inhibiting hypercholesterolemia is alirocumab. In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is evolocumab.

In some embodiments, the metabolic disorder is elevated liver enzymes (eg, ALT and/or AST) and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statins and fiber, or any combination thereof.

In some embodiments, the metabolic disorder is NASH and the therapeutic agent is obeticholic acid, seloncertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E, arachidylamidocolanoic acid, GS0976, emricasan, bolixivat , NGM282, GS9674, Tropexor, MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, Saroglitazar, BMS986036, ranifibranor, semaglutide, nitazoxanide, GRI_0621, EYP001, VK280 9, Nalmefen, LIK066 , MT_3995, elobisibat, namodenoson, poralumab, SAR425899, sotagliflozin, EDP_305, isosabutate, gemcabene, TERN_101, KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, namacizumab, CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, Celadepar, PXL770, TERN_201, NV556, AZD2693, SP_1373, VK0214, Hephastem, TGFTX4, RLBN1127 , GKT_137831, RYI_018, CB4209-CB4211, and JH_0920.

In some embodiments, the therapeutic agent treating the metabolic disorder is a melanocortin 4 receptor (MC4R) agonist. In some embodiments, MC4R agonists include proteins, peptides, nucleic acid molecules or small molecules. In some embodiments, the protein is a peptide analog of MC4R. In some embodiments, the peptide is setmelanotide. In some embodiments, the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. In some embodiments, the small molecule is 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid. In some embodiments, the MC4R agonist is ALB-127158(a).

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding predicted loss-of-function INHBE polypeptides; INHBE variant mRNA molecules encoding predicted loss-of-function INHBE polypeptides; or a therapeutic agent for treating or inhibiting cardiovascular disease for use in the treatment of cardiovascular disease in a subject having an INHBE variant cDNA molecule encoding a predicted loss-of-function INHBE polypeptide.

In some embodiments, the cardiovascular disease is hypertension and the therapeutic agent is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carte olol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quinapril hydrochloride, ramipril , trandolapril, candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, bepridil, diltiazem hydrochloride, felodipine, isradipine , nicardipine, nifedipine, nisoldipine, verapamil hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol labetalol hydrochloride, alpha methyldopa, clonidine hydrochloride, guanabenz acetate, guanfacine hydrochloride, guanadrel, guanethidine monosulfate, reserpine, hydralazine hydrochloride and minoxidil.

In some embodiments, the cardiovascular disease is cardiomyopathy, and the therapeutic agent is selected from: 1) antihypertensive agents such as ACE inhibitors, angiotensin II receptor blockers, beta blockers, and calcium channel blockers; 2) agents that slow the heart rate, such as beta blockers, calcium channel blockers and digoxin; 3) agents that keep the heart beating in a normal rhythm, such as antiarrhythmic agents; 4) electrolyte balancing agents such as aldosterone blockers; 5) Agents that remove excess fluid and sodium from the body, such as diuretics; 6) agents that prevent clot formation, such as anticoagulants or blood thinners; and 7) agents that reduce inflammation such as corticosteroids.

In some embodiments, the cardiovascular disease is heart failure and the therapeutic agent is selected from ACE inhibitors, angiotensin-2 receptor blockers, beta blockers, mineralocorticoid receptor antagonists, diuretics, ivabradine, sacubitril valsartan, hydralazine with nitrates, and digoxin. do.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding predicted loss-of-function INHBE polypeptides; INHBE variant mRNA molecules encoding predicted loss-of-function INHBE polypeptides; or an INHBE inhibitor that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having an INHBE variant cDNA molecule encoding a predicted loss-of-function INHBE polypeptide.

The present disclosure also relates to INHBE variant genomic nucleic acid molecules encoding predicted loss-of-function INHBE polypeptides; INHBE variant mRNA molecules encoding predicted loss-of-function INHBE polypeptides; or an INHBE inhibitor that treats or inhibits cardiovascular disease for use in the treatment of cardiovascular disease in a subject having an INHBE variant cDNA molecule encoding a predicted loss-of-function INHBE polypeptide.

In some embodiments, an INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. In some embodiments, an INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule. In some embodiments, the Cas protein is Cas9 or Cpf1. In some embodiments, the gRNA recognition sequence is located within SEQ ID NO:1. In some embodiments, the protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence. In some embodiments, a gRNA comprises about 17 to about 23 nucleotides. In some embodiments, the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27.

All patent documents, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, it means the version associated with the accession number on the effective filing date of the present application. Effective filing date means the earlier of the actual filing date or, where applicable, the filing date of the priority application referring to the accession number. Similarly, where different editions of publications, websites, etc. are published at different times, the most recently published version on the effective filing date of the application unless otherwise specified. Any feature, step, element, implementation or aspect of the invention may be used in combination with any other feature, step, element, implementation or aspect unless specifically indicated otherwise. Although the present invention has been described in some detail through examples and examples for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

The following examples are provided to further illustrate the implementations. They are intended to illustrate the claimed implementation and not to limit it. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and/or methods described herein are made and evaluated, and are intended to be purely illustrative and not limiting. All claims. Efforts have been made to ensure accuracy with respect to numbers (eg amounts, temperature, etc.) but some errors and deviations may be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or ambient temperature, and pressure is at or near atmospheric.

Example

Example 1: Loss of function in INHBE is associated with more favorable fat distribution and protection against type 2 diabetes in humans.

An exome-wide association analysis was performed on fat distribution as measured by waist-to-hip ratio adjusted for body mass index (BMI-adjusted WHR). BMI-adjusted WHR is a measure of body fat distribution independent of total adiposity. For each gene in the genome, an association with BMI-adjusted WHR for the burden of rare predicted loss-of-function genetic variants (pLOF variants with an alternative allele frequency [AAF] <1%) was estimated. In this analysis, the burden of rare predicted loss-of-function (pLOF) variants in INHBE (AAF<1%) was statistically significant at exome-wide levels (p<3.6x10 ^-7 , with Bonferroni correction for number tested). (i.e., lower WHR adjusted for BMI; see Figures 1 and 2). Table 6 shows the results of associations with fat distribution for pLOF variants in INHBE in 285,605 participants of European ancestry in the UKB cohort (association with BMI-adjusted WHR; genetic exposure burden of pLOF variants with AAF <1%). am). INHBE pLOF was strongly associated with low BMI-adjusted WHR (see Table 6). This statistically significant association was further recapitulated in a second tranche of UKB data (more than 140,000 participants of European ancestry) and a meta-analysis of additional data including more than 95,000 mixed-American participants from the MCPS study (see Figure 1). .

Table 6: INHBE gene-burden association results for BMI-adjusted WHR in UKB

Abbreviations: UKB = UK Biobank Study Population, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = number of individuals with no observation of heterozygous or homozygous pLOF variants in a gene, RA = at least one pLOF variant in a gene Number of individuals with heterozygous pLOF and no homozygous pLOF variant, AA = number of individuals with at least one homozygous pLOF variant in the gene, CI = confidence interval, pLOF = predicted loss-of-function, SD = standard deviation.

Table 6 shows the association of INHBE pLOF with BMI-adjusted-WHR in individuals of European ancestry in the UK Biobank study population. Effects of the INHBE pLOF variants were estimated in units of standard deviation (SD) and in units of the ratio of WHR. Table 6 shows that INHBE pLOF carriers had a lower BMI-adjusted WHR compared to the average of individuals without these genetic variants in analyzes adjusted for covariates, ancestry, and relatedness. Genotype counts are defined as cases of T2D in a cohort study, no variant leading to pLOF in INHBE (RR), one or more variants leading to pLOF in a single INHBE allele (RA), or one or more pLOF variants in both INHBE alleles (AA). Displays the number of objects that do not belong to the possessed T2D category.

This association of INHBE pLOF variants with low BMI-adjusted WHR was consistent in males and females in the UK Biobank cohort (see Table 7; genetic exposure is the burden of pLOF variants with AAF <1%).

Table 7: Sex-Stratified INHBE pLOF Variant Associations in UKB

Abbreviations: UKB = UK Biobank Study Population, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = number of individuals with no observation of heterozygous or homozygous pLOF variants in a gene, RA = at least one pLOF variant in a gene Number of individuals with heterozygous pLOF and no homozygous pLOF variant, AA = number of individuals with at least one homozygous pLOF variant in the gene, CI = confidence interval, pLOF = predicted loss-of-function, SD = standard deviation.

Table 7 shows the association of INHBE pLOF with BMI-adjusted WHR in individuals of European ancestry stratified by sex in the UK Biobank study. Effects of the INHBE pLOF variants were estimated in units of standard deviation (SD) and in units of the ratio of WHR. Genotype numbers are defined as individuals with no variant leading to pLOF of INHBE (RR) in a population study, or one or more variants leading to pLOF of a single INHBE allele (RA), or one or more pLOF variants in both INHBE alleles (AA). indicate the number The association of the INHBE pLOF variant with low BMI-adjusted WHR was similarly strong in men and women included in this analysis.

Among pLOF variants in INHBE, the variant most strongly associated with BMI-adjusted WHR was the c.299-1G>C (12:57456093:G:C according to GRCh38/hg38 human genome assembly coordinates) mutation, intron 1 receptor It was predicted to affect the splice site, shortening exon 2 by 12 nucleotides at the 5' end (see Figure 3 and Table 8) and leading to an in-frame deletion within the pro-domain of the INHBE protein (see Figure 4 ).

Table 8: Effect on splicing for 12:57456093:G:C receptor splice-site variants as predicted by 12:SpliceAI software

Delta score: A value between 0-1, interpreted as the probability of a variant having a splice-change effect on the INHBE gene.

Table 8 shows the predicted effect of variant 12:57456093:G:C on splicing of the INHBE gene.

The c.299-1G>C splice variant was expressed in Chinese hamster ovary (CHO) cells and was found to produce a low-molecular-weight protein that is not secreted outside the cell, indicating loss-of-function (FIG. 5).

In INHBE, pLOF variants were associated with greater hip circumference and higher arm and leg fat mass, suggesting the ability to store more calories in peripheral adipose tissue (see Figure 6 and Table 9).

Table 9: Association of pLOF Gene Variants in INHBE with Fatty Phenotype Meta-Analyzed in UKB, Geisinger Health System (GHS) and MCPS Studies

Abbreviations: UKB = UK Biobank Study Population, GHS = Geisinger Health System (GHS) Study Population, MCPS = Mexico City Prospective Study Population, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = number of individuals with no observation of heterozygous or homozygous pLOF variant in the gene, RA = number of individuals with at least one heterozygous pLOF in the gene and no homozygous pLOF variant, AA = at least one homozygous in the gene Number of individuals with pLOF variants, CI = confidence interval, pLOF = predicted loss-of-function, SD = standard deviation, kg/m ² = kilograms per square meter, cm = centimeters. Genotype numbers are defined as individuals who have no variant leading to pLOF of INHBE (RR) in a population study, one or more variants leading to pLOF of a single INHBE allele (RA), or one or more pLOF variants in both INHBE alleles (AA). indicate the number

Table 9 shows the association of INHBE pLOF with BMI, waist circumference and hip circumference. The effect of the INHBE pLOF is quantified in standard deviation units or the corresponding clinical unit for each anthropometric variable.

A rare pLOF variant in INHBE was also associated with protection against type 2 diabetes in humans. In addition, INHBE pLOF variants were found to be associated with a lower risk of type 2 diabetes (T2D) (see Table 10; genetic exposure burden of pLOF variants with AAF <1%), indicating that type 2 diabetes in humans. and constitute the first piece of evidence linking INHBE's LOF.

Table 10: Association of pLOF gene variants in T2D and INHBE in UKB, GHS and MCPS studies

Abbreviations: meta-analysis = joint analysis of all study populations listed, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = number of individuals with no observation of heterozygous or homozygous pLOF variants in a gene, RA = in a gene Number of individuals with at least one heterozygous pLOF and no homozygous pLOF variant, AA = number of individuals with at least one homozygous pLOF variant in the gene, CI = confidence interval, pLOF = predicted loss-of-function, SD = Standard Deviation. Genotype numbers are defined as individuals who have no variant leading to pLOF of INHBE (RR) in a population study, one or more variants leading to pLOF of a single INHBE allele (RA), or one or more pLOF variants in both INHBE alleles (AA). indicate the number

Table 10 shows associations with T2D for pLOF variants in INHBE in analyzes of the UK Biobank (UKB), Geisinger Health System (GHS) and Mexico City Prospective Study (MCPS) cohorts. The results show that within each study population, the INHBE pLOF variant is associated with a lower risk of T2D, which was confirmed in a meta-analysis combining results across all three study populations.

In addition, the INHBE pLOF variant was associated with favorable metabolic profiles in analyzes across multiple cohorts, including low HbA1c, ALT, triglycerides and LDL-C, and high HDL-C (see Table 11; genetic exposure indicated that AAF < burden of INHBE pLOF variants of 1%).

Table 11: Association of pLOF gene variants in INHBE with metabolic meta-analysis across UKB, GHS and MCPS studies

Abbreviations: UKB = UK Biobank Study Group, GHS = Geisinger Health System Study Group, MCPS = Mexico City Prospective Study Group, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = frequency of pLOF variants in a gene. Number of individuals with no observation of heterozygosity or homozygosity, RA = number of individuals with at least one heterozygous pLOF in the gene and no homozygous pLOF variant, AA = number of individuals with at least one homozygous pLOF variant in the gene , CI = confidence interval, pLOF = predicted loss-of-function, SD = standard deviation, mg/dL = milligrams per deciliter, U/L = units per liter. Genotype numbers are defined as individuals who have no variant leading to pLOF of INHBE (RR) in a population study, one or more variants leading to pLOF of a single INHBE allele (RA), or one or more pLOF variants in both INHBE alleles (AA). indicate the number

Table 11 shows the association of INHBE pLOF variants with metabolic phenotypic ranges estimated from the meta-analyses of the UKB, GHS and MCPS study populations. Results are expressed in units of standard deviation and original clinical units of relevant metabolic phenotypes.

In addition, INHBE pLOF variants were associated with reduced liver inflammation index on magnetic resonance imaging (see Table 12; genetic exposure burden of INHBE pLOF variants with AAF <1%).

Table 12: Association of pLOF gene variants with liver imaging phenotypes in INHBE at UKB

^a Adjusted for technical covariates including BMI, alcohol use and diabetes.

Abbreviations: PDFF = proton density fat fraction (defined as the ratio of the mobile proton density of fat (triglycerides) to the total density of protons from mobile triglycerides and mobile water, reflecting the concentration of fat in the tissue), ECF = extracellular fluid, T1 = longitudinal Time constant for magnetization recovery. It is the relaxation time that measures how quickly the net magnetization returns to its ground state. It can vary greatly depending on the strength of the magnetic field and tissue composition. It also increases with increasing magnetic field, whereas it decreases with fat and/or iron in the tissue. cT1 = T1 corrected for the effect of liver iron content resulting in an underestimation of the T1 value. UKB = UK Biobank Study Population, AAF = frequency of pLOF alleles across pLOF variants in a gene, RR = number of individuals with no observation of heterozygous or homozygous pLOF variants in a gene, RA = at least one heterozygous in a gene Number of individuals with pLOF and no homozygous pLOF variant, AA = number of individuals with at least one homozygous pLOF variant in the gene, CI = confidence interval, pLOF = predicted loss-of-function, SD = standard deviation.

Table 12 shows the association of INHBE pLOF variants with various liver imaging phenotypes in individuals of European ancestry from the UK Biobank study population. Results show that INHBE pLOF variants are associated with lower levels of ECF and cT1, which are measures of liver inflammation as defined by magnetic resonance imaging.

We further investigated whether INHBE pLOF variants were associated with liver histopathological phenotypes in 3565 bariatric surgery patients from the GHS cohort who underwent exome sequencing and perioperative liver wedge biopsy. Although there were only 3 carriers for the pLOF variant in the INHBE of that set, carrier status was assessed by a low NAFLD activity score (see Table 13), a liver disease severity scale on biopsy summing up steatosis, lobular inflammation, and swelling grades, and (Kleiner et al., Hepatology, 2005, 41, 1313-21).

Table 13: Association with low non-alcoholic fatty liver disease activity score for rare pLOF variants in INHBE

Associations with NAFLD activity scores (outcomes) for rare pLOF variants in INHBE have been reported. An association was estimated in 3,565 bariatric surgery patients in the GHS.

Finally, a common variant near INHBE (12:57259799:A:C; rs7966846; AAF, 0.28) was found to be associated with higher liver expression levels of INHBE mRNA (per allele beta, as part of bariatric surgery). 0.3 SD of INHBE transcript abundance quantified by RNASeq in more than 2,000 GHS participants who underwent liver biopsies). The 12:57259799:A:C variant was also found to be associated with higher BMI-adjusted WHR, triglycerides and type 2 diabetes risk. Higher expression of allele C resulted in higher BMI-adjusted WHR (p-value=1.5 x 10 ^-4 ), higher triglycerides (p-value=2.0 x 10 ^-11 ), and higher T2D risk (p-value = 0.03) (see Table 14). This shows that genetically-determined overexpression of INHBE is associated with a high risk of metabolic disease, while loss of function is associated with a favorable metabolic profile and low risk of diabetes (as noted above in association with pLOF variants).

Table 14: Association of INHBE eQTL, 12:57259799:A:C with various metabolic phenotypes in UKB and GHS cohorts

^a The estimate is the odds ratio.

Abbreviations: AAF = allele frequency of INHBE inter-expression enhancing alleles (i.e., alternative alleles), CI = confidence interval, SD = standard deviation, RR = reference-reference allele, RA = reference-alternative allele, AA = replacement-alternative allele, mg/dL = milligrams per deciliter. The genotype count is the number of individuals with no copy (RR), only one copy (RA), and two copies (AA) of the INHBE elevated allele in a population study. display Genotype numbers are further stratified within individuals classified as T2D cases in the study population.

Associations of 12:57259799:A:C with triglyceride levels, WHRadjBMI and risk of T2D were studied in all European ancestry participants from the UK Biobank and Geisinger Health Study. Results show that 12:57259799:A:C was significantly associated with higher triglyceride levels and higher BMI-adjusted WHR; It was also associated with a higher risk of T2D.

Example 2: INHBE is highly expressed in human hepatocytes and its expression is upregulated in patients with steatosis and non-alcoholic steatohepatitis

The mRNA expression of INHBE across human tissues was examined from the Genotype Tissue Expression Consortium (GTEx), and INHBE was found to be most expressed in the liver among GTEx tissues (see Fig. 7). Among the cell types, the mRNA expression of INHBE was also investigated in the data of the Human Protein Atlas (HPA), and as a result, INHBE was found to be most expressed in hepatocytes (see FIG. 7). The expression level of INHBE was also estimated in the livers of more than 2,000 bariatric surgery patients at GHS underwent liver RNASeq. It was found that INHBE expression was upregulated in patients with hepatic steatosis compared to subjects with normal livers, in patients with non-alcoholic steatohepatitis compared to subjects with normal livers, and in patients with non-alcoholic steatohepatitis compared to subjects with steatosis (see Figure 8). .

Example 3: Association with MRI-measured visceral to thigh fat ratio for INHBE identified in BMI-adjusted WHR finding analysis

A subset of the approximately 46,000 participants in the UKB used a Siemens MAGNETOM Aera 1.5T clinical MRI scanner for 2-point Dixon (Dixon, Radiology, 1984, 153, 189-194) subdivided into six different imaging series. ) underwent MRI (Littlejohns et al., Nat. Commun., 2020, 11, 2624). This subset included 38,880 individuals with available exome sequencing. Stitching of six different scan locations was corrected similarly to what was previously done for overlapping slices, partial scans, repeat scans, fat-water exchange, misalignment between imaging series, bias-fields, artificially dark slices and local hotspots ( Basty et al., Image Processing and Quality Control for Abdominal Magnetic Resonance Imaging in the UK Biobank, 2020, ArXiv abs/2007.01251). A total of 52 subjects were manually annotated by whole-body Dickson MRI into six fat grades: upper body fat, abdominal fat, visceral fat, mediastinum fat, thigh fat, and lower thigh fat. PPARG (Ludtke et al., J. Med. Genet., 2007, 44, e88), PLIN1 (Gandotra et al., N. Engl. J. Med., 2011, 364, 740-748), LMNA (Jeru et al. al., J. Med. Genet., 2017, 54, 413-416), LIPE (Zolotov et al., Am. J. Med. Genet., 2017, A 173, 190-194) and MC4R (Akbari et al. Special care was taken to tailor the training data set to attempt to encompass the expected phenotypic variability by specifically including training subjects with genetic mutations predisposing them to abnormal fat and muscle phenotypes, such as ., Science, 2021, 373). tilted Using these annotations, UNet (Weng et al., IEEE Access, 2021, 9, 16591-16603) architecture and ResNet34 (He et al., in 2016 IEEE Conference on Computer Vision and Pattern Recognition (CVPR), 2016, 770- 778) trained a multi-class segmentation deep neural network using backbone, and loss of function of the Jaccard index and categorical defocus sum (Lin et al., IEEE Transactions on Pattern Analysis and Machine Intelligence, 2020, 42, 318 -327). The fat volume phenotype was calculated by summing the segmentation map results of the neural network for each corresponding fat class. Visceral-to-femoral fat ratio was calculated as the ratio of visceral-to-femoral fat volume for a given individual.

Rare coding variants in INHBE associated with BMI-adjusted WHR were found in a subset of 38,880 individuals who underwent whole-body MRI at UKB (i.e., ~6% of the discovery sample), a refined measure of fat distribution, the visceral-to-femoral fat ratio on MRI. showed a very consistent association with (see Table 15). In the subset of UKB with MRI data, there was a nominally significant association with low MRI-defined visceral-to-femoral fat ratio for the INHBE pLOF variant (beta in SD units of fat ratio per allele, -0.24; 95% CI; - 0.45 to -0.02, p = 0.03, see Table 15).

Table 15

Each gene-burden outcome in the table was analyzed in a model describing the sex-specific effects of age, body mass index, and height on visceral-to-thigh fat ratio.

Abbreviations: pLOF, predicted loss of function; AAF, alternative allele frequency; CI, confidence interval; SD, standard deviation; BMI, body mass index; p, P-value; RR, reference homozygous genotype; RA, reference-alternative genotype; AA, alternative homozygous genotype.

Example 4: INHBE predicted loss-of-function association with increased left ventricular ejection fraction and protection from cardiomyopathy

Cases in the present example were all study participants without heart disease. Results were based on a meta-analysis of UKB, GHS, SINAI, UPENN-PMBB, MDCS, Indiana-Chalasani. The predicted loss-of-function of INHBE associated with increased left ventricular ejection fraction and protection from cardiomyopathy is shown in Table 16 (burden of INHBE rare pLoF variant (M1.1)).

Table 16

Result 1 is the left ventricular ejection fraction ^* .

Outcome 2 is non-ischemic cardiomyopathy ^** .

^* Left ventricular ejection fraction obtained from cardiac MRI of UK Biobank participants.

^** Non-ischemic cardiomyopathy cases were defined as study participants with one or more of the following ICD10 codes: I420 (dilated cardiomyopathy), I425 (other restrictive cardiomyopathy), I428 (other non-compressive cardiomyopathy), I429 (other Primary cardiomyopathy|unspecified), and absence of one or more of any ICD10 codes indicative of myocardial infarction (I21|I22|I23|I252|I256) and hypertrophic cardiomyopathy (I421, I422).

The association of pLOF variants with lower blood pressure (see Table 17; INHBE rare pLOF variant burden - M1.1) is consistent with a beneficial effect on hemodynamic properties.

Table 17

Characteristic 1 is diastolic blood pressure (corrective treatment).

Characteristic 2 is systolic blood pressure (corrective treatment).

Various modifications of the described subject matter other than those described herein will be apparent to those skilled in the art from the foregoing description. Such variations are also intended to be within the scope of the appended claims. Each reference cited in this application (including but not limited to journal articles, US and non-US patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is complete and is hereby incorporated for all purposes. included by reference.

SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. 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cccaggcagc 420 gcugaccaga gcccuccgga gacuacagcc agggagugug gcuccaggga auggggagga 480 ggucaucagc uuugcuacug ucacagacuc cacuucagcc uacagcuccc ugcucacuuu 540 ucaccugucc acuccucggu cccaccaccu guaccaugcc cgccuguggc ugcacgugcu 600 ccccacccuu ccuggcacuc uuugcuugag gaucuuccga uggggaccaa ggaggaggcg 660 ccaagggucc cgcacucucc uggcugagca ccacaucacc aaccugggcu ggcauaccuu 720 aacucugccc ucuaguggcu ugagggguga gaagucuggu guccugaaac ug caacuaga 780 cugcagaccc cuagaaggca acagcacagu uacuggacaa ccgaggcggc ucuuggacac 840 agcaggacac cagcagcccu uccuagagcu uaagauccga gccaaugagc cuggagcagg 900 ccgggccagg aggaggaccc ccaccuguga gccugcgacc cccuuauguu gcaggc gaga 960 ccauuacgua gacuuccagg aacuggggaug gcgggacugg auacugcagc ccgaggggua 1020 ccagcugaau uacugcagug ggcagugccc uccccaccug gcuggcagcc caggcauugc 1080 ugccucuuuc cauucugccg ucuucagccu ccucaaagcc aacaauccuu ggccugccag 1140 uaccuccugu uguguccua cugcccgaag gccccucucu cuccucuacc uggaucau aa 1200 uggcaaugug gucaagacgg augugccaga uaugguggug gaggccugug gcugcagcua 1260 gcaagcagga ccuggggcuu uggagugaag agaccaagau gaaguuuccc aggcacaggg 1320 caucugugac uggaggcauc agauuccuga uccacacccc aacccacaa ccac cuggca 1380 auaugacuca cuugaccccu auggggaccca aaugggcacu uucuugucug agacucuggc 1440 uuauuccagg uuggcugaug uguugggaga uggguaaagc guuucuucua aaggggucua 1500 cucagaaagc augauuuccu gcccuaaguc cugugagaag augucaggga cuagggaggg 1560 agggagggaa ggcagagaaa aauuacuuag ccucucccaa gaugagaaag uccucaagug 1620 agggggaggag gaagcagaua gaugguccag caggcuugaa gcaggguaag caggcuggcc 1680 caggguaagg gcuguugagg uaccuuaagg gaaggucaag agggagaugg gcaaggcgcu 1740 gagggaggau gcuuagggga cccccagaaa caggagucag gaaaaugagg cacuaagccu 1800 aagaaguucc cugguuuuuc ccaggggaca ggacccacug ggcgacaagc auuuauacuu 1860 ucuuucuucu 20 40 gagguuucaa cauguuggcc aggauggucu caaucucuug accucuugau ccacccgacu 2100 uggccucccg aagugaugag auuauaggcg ugagccaccg cgccuggcuu auacuuucuu 2160 aauaaaaagg agaaagaaaa ucaacaaaug ugagucauaa agaaggguua ggguga uggu 2220 ccagagcaac aguucuucaa guguacucug uaggcuucug ggaggucccu uuucaggggu 2280 guccacaaag ucaaagcuau uuucauaaua auacuaacau guuauuugcc uuuugaauuc 2340 ucauuaucuu aaaauuguau uguggaguuu uccaaaggcc gugugacaug ugauuacauc 2400 aucuuucuga caucaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa aaaa 2459 <210> 5 <211> 1616 <212> DNA <213> homo sapien <400> 5 atgagctgg agggtcaagc acagctatcc atcagatgat ctactttcag ccttcctgag 60 tcccagacaa tagaagacag gtggctgtac ccttggccaa gggtaggtgt ggcagtggtg 120 tctgctgtca ctgtgccctc attggccccc agcaatcaga ctcaacagac ggagcaactg 180 ccatccgagg ctcctgaacc agggccattc accaggagca tgcggctccc tgatgtccag 240 ctctggctgg tgctgctgtg c gctgaccag agccctccgg agactacagc cagggagtgt ggctccaggg 480 aatggggagg aggtcatcag ctttgctact gtcacagact ccacttcagc ctacagctcc 540 ctgctcactt ttcacctgtc cactcctcgg tcccaccacc tgtaccatgc ccgcctgtgg 600 ctgcacgtgc tccccaccct tcctggcact ctttgcttga ggatcttccg atggggacca 660 aggaggaggc gccaagggtc ccgcactctc ctggctgagc accacatcac caacctgggc 720 tggcatacct taactctgcc ctctagtggc ttgaggggtg agaagtctgg tgtcctgaaa 780 ctgcaactag actgcagacc cctagaaggc aacagcacag ttactggaca accgaggcgg 840 ctcttggaca cagcaggaca ccagcagccc ttcctagagc ttaagatccg agccaatgag 900 cctggagcag gccgggccag gaggaggacc cccacctgtg agcctgcgac ccccttatgt 960 tgcaggcgag accattacgt agacttccag gaactgggat ggcgggactg gatactgcag 1020 cccgaggggt accagctgaa ttactgcagt gggcagtgcc ctccccacct ggctggcagc 1080 ccaggcattg ctgcctcttt ccattctgcc gtcttcagcc t cctcaaagc caacaatcct 1140 tggcctgcca gtacctcctg ttgtgtccct actgcccgaa ggcccctctc tctcctctac 1200 ctggatcata atggcaatgt ggtcaagacg gatgtgccag atatggtggt ggaggcctgt 1260 ggctgcagct agcaagagga cctggggctt tggagtgaag agaccaagat gaagtttccc 1320 aggcacaggg catctgtgac tggaggcatc agattcctga tccacacccc aacccaacaa 1380 ccacctggca atatgactca cttgacccct atgggaccca aatgggcact ttcttgtctg 1440 agactctggc ttattccagg ttggctgatg tgttgggaga tgggtaaagc gtttcttcta 1500 aaggggtcta cccagaaagc atgatttcct gccctaagtc ctgtgagaag atgtcaggga 1560 ctagggaggg agggagggaa ggcagagaaa aattacttag cctctcccaa gatgag 1616 <210> 6 <211> 2445 <212> DNA <213> homo sapien <400> 6 agctgtgagg gtcaagcaca gctatccatc agatgatcta ctttcagcct tcctgagtcc 60 cagacaatag aagacaggtg gctgtaccct tggccaaggg taggtgtggc agtggtgtct 120 gctgtcactg tgccctcatt ggcccccagc aatcagactc aacagacgga gcaactgcca 1 80 tccgaggctc ctgaaccagg gccattcacc aggagcatgc ggctccctga tgtccagctc 240 tggctggtgc tgctgtgggc actggtgcga gcacagggga cagggtctgt gtgtccctcc 300 tgtgggggct ccaaactggc accccaagca gaacgagctc tggtgctgga gctag ccaag 360 cagcaaatcc tggatgggtt gcacctgacc agtcgtccca gaataactca tcctccaccc 420 caggcagcgc tgaccagagc cctccggaga ctacagccag ggaggtgtggc tccagggaat 480 ggggaggagg tcatcagctt tgctactgtc acagactcca cttcagccta cagctccctg 540 ctcacttttc acctgtccac tcctcggtcc caccacctgt accatgcccg cctgtggctg 600 cac gtgctcc ccacccttcc tggcactctt tgcttgagga tcttccgatg gggaccaagg 660 agggaggcgcc aagggtcccg cactctcctg gctgagcacc acatcaccaa cctgggctgg 720 cataccttaa ctctgccctc tagtggcttg aggggtgaga agtctggtgt cctgaaactg 7 80 caactagact gcagacccct agaaggcaac agcacagtta ctggacaacc gaggcggctc 840 ttggacacag caggacacca gcagcccttc ctagagctta agatccgagc caatgagcct 900 ggagcaggcc gggccaggag gaggaccccc acctgtgagc ctgcgacccc cttatgttgc 960 aggcgagacc attacgtaga cttccaggaa ctgggatggc gggactggat actgcagccc 1020 gaggggtacc agct cctctacctg 1200 gatcataatg gcaatgtggt caagacggat gtgccagata tggtggtgga ggcctgtggc 1260 tgcagctagc aagaggacct ggggctttgg agtgaagaga ccaagatgaa gtttcccagg 1320 cacagggcat ctgtgactgg aggcatcaga ttcctgatcc acaccccaac ccaacaacca 1380 cctggcaata tgactcactt gacccctatg ggacccaaat gggcactttc ttgtctgaga 1440 ctctggctta ttccaggttg gctga tgtgt tgggagatgg gtaaagcgtt tcttctaaag 1500 gggtctaccc agaaagcatg atttcctgcc ctaagtcctg tgagaagatg tcagggacta 1560 gggagggagg gagggaaggc agagaaaaat tacttagcct ctcccaagat gagaaagtcc 1620 tcaagt gagg ggaggaggaa gcagatagat ggtccagcag gcttgaagca gggtaagcag 1680 gctggcccag ggtaagggct gttgaggtac cttaagggaa ggtcaagagg gagatgggca 1740 aggcgctgag ggaggatgct taggggaccc ccagaaacag gagtcaggaa aatgaggcac 1800 taagcctaag aagttccctg gtttttccca ggggacagga cccactggga gacaagcatt 1860 tatactttct ttcttctttt ttattttt tt gagatcgagt ctcgctctgt caccaggctg 1920 gagtgcagtg acacgatctt ggctcactgc aacctccgtc tcctgggttc aagtgattct 1980 tctgcctcag cctcccgagc agctgggatt acaggcgccc actaattttt gtattcttag 2040 tagaaacgag gt ttcaacat gttggccagg atggtctcaa tctcttgacc tcttgatcca 2100 cccgacttgg cctcccgaag tgatgagatt ataggcgtga gccaccgcgc ctggcttata 2160 ctttcttaat aaaaaggaga aagaaaatca acaaatgtga gtcataaaga agggttaggg 2220 tgatggtcca gagcaacagt tcttcaagtg tactctgtag gcttctggga ggtccctttt 2280 caggggtgtc cacaaagtca aagctatttt cataataata c taacatgtt atttgccttt 2340 tgaattctca ttatcttaaa attgtattgt ggagttttcc agaggccgtg tgacatgtga 2400 ttacatcatc tttctgacat cattgttaaa aaaaaaaaaa aaaaa 2445 <210> 7 <211> 2459 <212> DNA <213> homo sapien <400> 7 gggtcaagca cagctatcca tcagatgatc tactttcagc cttcctgagt cccagacaat 60 agaagacagg tggctgtacc cttggccaag ggtaggtgtg gcagtggtgt ctgctgtcac 120 tgtgccctca ttggccccca gcaatcagac tcaacagacg gagcaactgc catccgaggc 180 tcctgaacca gggccattca ccaggagcat gcggctccct gatgtccagc tctggctggt 240 gctgctgtgg gcactggtgc gagcacaggg gacagggtct gtgtgtccct cctgtggggg 300 ctccaaactg gcaccccaag cagaacgagc tctggtgctg gagctagcca agcagcaaat 360 cctggatggg ttgcacctga ccagtcgtcc cagaataact catcctccac cccaggcagc 420 gctgacc aga gccctccgga gactacagcc agggagtgg gctccaggga atggggagga 480 ggtcatcagc tttgctactg tcacagactc cacttcagcc tacagctccc tgctcacttt 540 tcacctgtcc actcctcggt cccacccacct gtaccatgcc cgcctgtggc tgcacgtgct 600 ccccaccctt cctggcactc tttgcttgag gatcttccga tggggaccaa ggaggaggcg 660 ccaagggtcc cgcactctcc cag cagccct tcctagagct taagatccga gccaatgagc ctggagcagg 900 ccgggccagg aggaggaccc ccacctgtga gcctgcgacc cccttatgtt gcaggcgaga 960 ccattacgta gacttccagg aactgggatg gcgggactgg atactgcagc ccgaggggta 1020 ccagctgaat t actgcagtg ggcagtgccc tccccacctg gctggcagcc caggcattgc 1080 tgcctctttc cattctgccg tcttcagcct cctcaaagcc aacaatcctt ggcctgccag 1140 tacctcctgt tgtgtcccta ctgcccgaag gcccctctct ctcctctacc tggatcataa 1200 tggcaatgtg gtcaagacgg atgtgccaga tatggtggtg gaggcctgtg gctgcagcta 1260 gcaagcagga cctgggg ctt tggagtgaag agaccaagat gaagtttccc aggcacaggg 1320 catctgtgac tggaggcatc agattcctga tccacacccc aacccaacaa ccacctggca 1380 atatgactca cttgacccct atgggaccca aatgggcact ttcttgtctg agactctggc 1440 ttatccca gg ttggctgatg tgttgggaga tgggtaaagc gtttcttcta aaggggtcta 1500 ctcagaaagc atgatttcct gccctaagtc ctgtgagaag atgtcaggga ctagggaggg 1560 agggagggaa ggcagagaaa aattacttag cctctcccaa gatgagaaag tcctcaagtg 1620 agggggaggag gaagcagata gatggtccag caggcttgaa gcagggtaag caggctggcc 1680 cagggtaagg gctgttgagg taccttaagg gaaggtcaag agggagatgg gcaaggcgct 1740 gagggaggat gcttagggga cccccagaaa caggagtcag gaaaatgagg cactaagcct 1800 aagaagttcc ctggtttttc ccaggggaca ggacccactg ggcgacaagc atttatactt 1860 tctttcttct tttttatttt tt tgagatcg agtctcgctc tgtcaccagg ctggagtgca 1920 gtgacacgat cttggctcac tgcaacctcc gtctcctggg ttcaagtgat tcttctgcct 1980 cagcctcccg agcagctggg attacaggcg cccactaatt tttgtattct tagtagaaac 2040 gaggtttcaa catgttggcc aggatggtct caatctcttg acctcttgat ccacccgact 2100 tggcctcccg aagtgatgag attataggcg tga gccaccg cgcctggctt atactttctt 2160 aataaaaagg agaaagaaaa tcaacaaatg tgagtcataa agaagggtta gggtgatggt 2220 ccagagcaac agttcttcaa gtgtactctg taggcttctg ggaggtccct tttcaggggt 2280 gtccacaaag tcaaagct at tttcataata atactaacat gttatttgcc ttttgaattc 2340 tcattatctt aaaattgtat tgtggagttt tccaaaggcc gtgtgacatg tgattacatc 2400 atctttctga catcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa aaaaaaaaa 2459 <210> 8 <211> 350 <212> PRT <213> homo sapien <400> 8 Met Arg Leu Pro Asp Val Gln Leu Trp Leu Val Le u Leu Trp Ala Leu 1 5 10 15 Val Arg Ala Gln Gly Thr Gly Ser Val Cys Pro Ser Cys Gly Gly Ser 20 25 30 Lys Leu Ala Pro Gln Ala Glu Arg Ala Leu Val Leu Glu Leu Ala Lys 35 40 45 Gln Gln Ile Leu Asp Gly Leu His Leu Thr Ser Arg Pro Arg Ile Thr 50 55 60 His Pro Pro Pro Gln Ala Ala Leu Thr Arg Ala Leu Arg Arg Leu Gln 65 70 75 80 Pro Gly Ser Val Ala Pro Gly Asn Gly Glu Glu Val Ile Ser Phe Ala 85 90 95 Thr Val Thr Asp Ser Thr Ser Ala Tyr Ser Ser Leu Leu Thr Phe His 100 105 110 Leu Ser Thr Pro Arg Ser His His Leu Tyr His Ala Arg Leu Trp Leu 115 120 125 His Val Leu Pro Thr Leu Pro Gly Thr Leu Cys Leu Arg Ile Phe Arg 130 135 140 Trp Gly Pro Arg Arg Arg Arg Gln Gly Ser Arg Thr Leu Leu Ala Glu 145 150 155 160 His Ile Thr Asn Leu Gly Trp His Thr Leu Thr Leu Pro Ser Ser 165 170 175 Gly Leu Arg Gly Glu Lys Ser Gly Val Leu Lys Leu Gln Leu Asp Cys 180 185 190 Arg Pro Leu Glu Gly Asn Ser Thr Val Thr Gly Gln Pro Arg Arg Leu 195 200 205 Leu Asp Thr Ala Gly His Gln Gln Pro Phe Leu Glu Leu Lys Ile Arg 210 215 220 Ala Asn Glu Pro Gly Ala Gly Arg Ala Arg Arg Arg Thr Pro Thr Cys 225 230 235 240 Glu Pro Ala Thr Pro Leu Cys Cys Arg Arg Asp His Tyr Val Asp Phe 245 250 255 Gln Glu Leu Gly Trp Arg Asp Trp Ile Leu Gln Pro Glu Gly Tyr Gln 260 265 270 Leu Asn Tyr Cys Ser Gly Gln Cys Pro Pro His Leu Ala Gly Ser Pro 275 280 285 Gly Ile Ala Ala Ser Phe His Ser Ala Val Phe Ser Leu Leu Lys Ala 290 295 300 Asn Asn Pro Trp Pro Ala Ser Thr Ser Cys Cys Val Pro Thr Ala Arg 305 310 315 320 Arg Pro Leu Ser Leu Leu Tyr Leu Asp His Asn Gly Asn Val Val Lys 325 330 335 Thr Asp Val Pro Asp Met Val Val Glu Ala Cys Gly Cys Ser 340 345 350 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 9 cgtctgttga gtctgattgc 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 10 gacggagcaa ctgccatccg 20 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 11 atcagggagc cgcatgctcc 20 < 210> 12 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 12 ctgaaccagg gccattcacc 20 <210> 13 <211> 20 <212> DNA <213> artificial sequence < 220> <223> gRNA Recognition Sequence <400> 13 cctggttcag gagcctcgga 20 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 14 catccgaggc tcctgaacca 20 <210 > 15 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 15 ccatccgagg ctcctgaacc 20 <210> 16 <211> 20 <212> DNA <213> artificial sequence <220 > <223> gRNA Recognition Sequence <400> 16 gccacctgtc ttctattgtc 20 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 17 agccgcatgc tcctggtgaa 20 <210> 18 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 18 gtctgttgag tctgattgct 20 <210> 19 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 19 aagacaggtg gctgtaccct 20 <210> 20 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 20 ctgattgctg ggggccaatg 20 <210> 21 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 21 tgattgctgg gggccaatga 20 <210> 22 <211> 20 <212> DNA <213> artificial sequence <220> < 223> gRNA Recognition Sequence <400> 22 ccacctgtct tctattgtct 20 <210> 23 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 23 atgctcctgg tgaatggccc 20 <210> 24 < 211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 24 ctgttgagtc tgattgctgg 20 <210> 25 <211> 20 <212> DNA <213> artificial sequence <220> <223 > gRNA Recognition Sequence <400> 25 ctggtgaatg gccctggttc 20 <210> 26 <211> 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 26 accactgcca cacctaccct 20 <210> 27 <211 > 20 <212> DNA <213> artificial sequence <220> <223> gRNA Recognition Sequence <400> 27 tctgttgagt ctgattgctg 20 <210> 28 <211> 23 <212> DNA <213> artificial sequence <220> <223> original exon 2 <400> 28 agactccact tcagcctaca gct 23 <210> 29 <211> 23 <212> DNA <213> artificial sequence <220> <223> predicted exon 2<400> 29 acactccact tcagcctaca gct 23

Claims (160)

A method of treating a subject having or at risk of developing a metabolic disorder, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing type 2 diabetes comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing obesity, the method comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing elevated triglyceride levels, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing lipodystrophy, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing liver inflammation, the method comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing fatty liver disease, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing hypercholesterolemia, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing elevated liver enzymes, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing nonalcoholic steatohepatitis (NASH), comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing a cardiovascular disease, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing cardiomyopathy, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing hypertension, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. A method of treating a subject having or at risk of developing heart failure, comprising administering to the subject an inhibin subunit beta E (INHBE) inhibitor. 15. The method of any one of claims 1 to 14, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA) or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. 15. The method of any one of claims 1 to 14, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence in an INHBE genomic nucleic acid molecule. The method of claim 16, wherein the Cas protein is Cas9 or Cpf1. 17. The method according to claim 15 or 16, wherein the gRNA recognition sequence is located within SEQ ID NO: 1. 17. The method of claim 15 or 16, wherein the protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence. 20. The method of any one of claims 16-19, wherein the gRNA comprises from about 17 to about 23 nucleotides. 20. The method of any one of claims 16 to 19, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27. 22. The method of any one of claims 1-21, further comprising detecting the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample from the subject. 23. The method of claim 22, wherein if the subject is on INHBE criteria, the subject is also administered a therapeutic agent that treats or inhibits a metabolic disorder or cardiovascular disease at a standard dosage. 23. The method of claim 22, wherein if the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, the subject also has a metabolic disorder or A method of receiving a therapeutic agent that treats or inhibits cardiovascular disease. 25. The method of any one of claims 22 to 24, wherein the INHBE variant nucleic acid molecule is a missense variant, a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, or An in-frame indel variant, or a variant encoding a truncated INHBE polypeptide. 26. The method of claim 25, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide. 27. The method according to any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule. 28. The method of claim 27, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule in the biological sample. 27. The method according to any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is an mRNA molecule. 30. The method of claim 29, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample. 27. The method according to any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is a cDNA molecule generated from an mRNA molecule. 32. The method of claim 31, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of an INHBE cDNA molecule generated from mRNA molecules in the biological sample. 33. The method of any one of claims 22-32, wherein the detecting step comprises sequencing the entire nucleic acid molecule. A method of treating a subject suffering from a metabolic disorder with a therapeutic agent that treats or inhibits the metabolic disorder, the method comprising:
obtaining or obtaining a biological sample from a subject;
By performing or performing a genotyping assay on a biological sample to determine whether a subject has a genotype comprising an inhibin subunit beta E (INHBE) variant nucleic acid molecule
determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; and
If the subject is on INHBE criteria, administering or continuing administration to the subject at a standard dose of a therapeutic agent that treats or inhibits the metabolic disorder, and administering an INHBE inhibitor to the subject;
If the subject is heterozygous for the INHBE variant nucleic acid molecule, administering or continuing to administer to the subject a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or lower than the standard dose, and administering an INHBE inhibitor to the subject;
If the subject is homozygous for the INHBE variant nucleic acid molecule, administering to the subject or continuing to administer a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or lower than the standard dose;
wherein the presence of a genotype having an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing a metabolic disorder. 35. The method of claim 34, wherein the subject is on INHBE basis, and the subject receives or continues to receive standard doses of a therapeutic agent that treats or inhibits the metabolic disorder, and is administered an INHBE inhibitor. 35. The method of claim 34, wherein the subject is heterozygous for the INHBE variant nucleic acid molecule, and the subject receives or continues to receive a standard dose of a therapeutic agent that treats or inhibits a metabolic disorder in an amount equal to or lower than a standard dose and receives an INHBE inhibitor , method. A method of treating a subject suffering from cardiovascular disease with a therapeutic agent that treats or inhibits cardiovascular disease, the method comprising:
obtaining or obtaining a biological sample from a subject;
By performing or performing a genotyping assay on a biological sample to determine whether a subject has a genotype comprising an inhibin subunit beta E (INHBE) variant nucleic acid molecule
determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; and
When the subject is on INHBE criteria, a therapeutic agent for treating or inhibiting cardiovascular disease is administered to the subject at a standard dose or continues to be administered, and an INHBE inhibitor is administered to the subject;
If the subject is heterozygous for the INHBE variant nucleic acid molecule, a therapeutic agent that treats or inhibits cardiovascular disease is administered to the subject in an amount equal to or lower than the standard dose or continues to be administered, and an INHBE inhibitor is administered to the subject;
If the subject is homozygous for the INHBE variant nucleic acid molecule, administering to the subject or continuing to administer a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or lower than the standard dose;
The method of claim 1 , wherein the presence of a genotype having an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide indicates that the subject has a reduced risk of developing cardiovascular disease. 38. The method of claim 37, wherein the subject is on an INHBE basis, and the subject receives or continues to receive a standard dose of a therapeutic agent that treats or inhibits a cardiovascular disease, and is administered an INHBE inhibitor. 38. The method of claim 37, wherein the subject is heterozygous for the INHBE variant nucleic acid molecule, the subject receives or continues to receive a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or lower than a standard dose, and administers an INHBE inhibitor. how to receive. 38. The method of claim 34 or 37, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule. 41. The method of claim 40, wherein the genotyping comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule in the biological sample. 38. The method according to claim 34 or 37, wherein the INHBE variant nucleic acid molecule is an mRNA molecule. 43. The method of claim 42, wherein the genotyping comprises sequencing at least a portion of the nucleotide sequence of an INHBE mRNA molecule in a biological sample. 38. The method of claim 34 or 37, wherein the INHBE variant nucleic acid molecule is a cDNA molecule generated from an mRNA molecule. 45. The method of claim 44, wherein the genotyping comprises sequencing at least a portion of the nucleotide sequence of INHBE cDNA molecules generated from mRNA molecules in the biological sample. 38. The method of claim 34 or 37, wherein the INHBE variant nucleic acid molecule is a missense variant, a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, or an in-frame indel. variant, or variant encoding a truncated INHBE polypeptide. 38. The method of claim 34 or 37, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide. 48. The method of any one of claims 34-47, wherein the genotyping assay comprises sequencing total nucleic acid molecules in a biological sample. 49. The method of any one of claims 34 to 48, wherein the genotyping assay
a) amplifying at least a portion of a nucleic acid molecule encoding an INHBE polypeptide;
b) labeling the amplified nucleic acid molecule with a detectable label;
c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and
d) detecting the detectable label. 50. The method of claim 49, wherein the nucleic acid molecule in the sample is an mRNA and the mRNA is reverse transcribed into cDNA prior to the amplification step. 49. The method of any one of claims 34 to 48, wherein the genotyping assay
contacting a nucleic acid molecule in a biological sample with an alteration-specific probe comprising a detectable label; and
detecting the detectable label. 52. The method of any one of claims 34-51, wherein the nucleic acid molecule is present in a cell obtained from the subject. 53. The method of any one of claims 34-52, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. 53. The method of any one of claims 34-52, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule. 55. The method of claim 54, wherein the Cas protein is Cas9 or Cpf1. 56. The method of claim 54 or 55, wherein the gRNA recognition sequence is located within SEQ ID NO: 1. 56. The method of claim 54 or 55, wherein the protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence. 58. The method of any one of claims 54-57, wherein the gRNA comprises from about 17 to about 23 nucleotides. 58. The method of any one of claims 54-57, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27. 37. The method of any one of claims 34 to 36, wherein the metabolic disorder is type 2 diabetes, and the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, thiazoli Dindione, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, semaglutide, canagliflozin, dapagliflozin and empagliflozin or these selected from any combination of 37. The method of any one of claims 34 to 36, wherein the metabolic disorder is obesity and the treatment is selected from orlistat, phentermine, topiramate, bupropion, naltrexone and liraglutide or any combination thereof. . 37. The method of any one of claims 34 to 36, wherein the metabolic disorder is elevated triglycerides, and the therapeutic agent is rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil and fenofibric acid, niacin and omega-3 fatty acids, or selected from any combination thereof. 37. The method according to any one of claims 34 to 36, wherein the metabolic disorder is lipodystrophy, and the therapeutic agent is tesamorelin, metformin, poly-L-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, selected from human collagen, silicone, and hyaluronic acid or any combination thereof. 37. The method according to any one of claims 34 to 36, wherein the metabolic disorder is liver inflammation and the therapeutic agent is a hepatitis treatment or hepatitis vaccine. 37. The method according to any one of claims 34 to 36, wherein the metabolic disorder is fatty liver disease and the subject undergoes bariatric surgery and/or dietary intervention. 37. The method according to any one of claims 34 to 36, wherein the metabolic disorder is hypercholesterolemia, and the therapeutic agent is atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, selected from Colesevelam and Colestipol, Alirocumab, Evolocumab, Niaspan, Niacor, Fenofibrate, Gemfibrozil, and Bemfedoic or any combination thereof. 37. The method of any one of claims 34 to 36, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statins and fiber, or any combination thereof. . 37. The method of any one of claims 34-36, wherein the metabolic disorder is non-alcoholic steatohepatitis (NASH), and the therapeutic agent is obeticholic acid, seloncertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E , arachidylamidocolanoic acid, GS0976, emricasan, bolixivat, NGM282, GS9674, tropexor, MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, saroglitazar, BMS986036, ranifibranor, sema Glutide, nitazoxanide, GRI_0621, EYP001, VK2809, nalmefene, LIK066, MT_3995, elobisibat, namodenoson, poralumab, SAR425899, sotagliflozin, EDP_305, isosabutate, gemcabene, TERN_101 , KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, Namacizumab, CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, Sella Depar, PXL770, TERN_201, NV556, AZD2693, SP_1373, VK0214, Hephastem, TGFTX4, RLBN1127, GKT_137831, RYI_018, CB4209-CB4211, and JH_0920. 37. The method of any one of claims 34-36, wherein the therapeutic agent for treating or inhibiting a metabolic disorder is a melanocortin 4 receptor (MC4R) agonist. 70. The method of claim 69, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule or small molecule. 70. The method of claim 69, wherein the protein is a peptide analog of MC4R. 72. The method of claim 71, wherein the peptide is setmelanotide. 70. The method of claim 69, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. 71. The method of claim 70, wherein the small molecule is 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid. 70. The method of claim 69, wherein the MC4R agonist is ALB-127158(a). 40. The method according to any one of claims 37 to 39, wherein the cardiovascular disease is hypertension, and the therapeutic agent is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol , betaxolol, bisoprolol fumarate, carteolol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, mo exipril, perindopril, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, bepridil , diltiazem hydrochloride, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, verapamil hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol labetalol hydrochloride, alpha methyldopa, clonidine hydrochloride, guanabenz acetate, guanfacine hydrochloride, guanadrel, guanethidine monosulfate, reserpine, hydralazine hydrochloride and minoxidil or any combination thereof. 40. The method according to any one of claims 37 to 39, wherein the cardiovascular disease is cardiomyopathy, and the therapeutic agent is an ACE inhibitor, an angiotensin II receptor blocker, a beta blocker, a calcium channel blocker, digoxin, an antiarrhythmic agent, an aldosterone blocker, a diuretic, anticoagulants, blood thinners and corticosteroids. 40. The method according to any one of claims 37 to 39, wherein the cardiovascular disease is heart failure, and the therapeutic agent is an ACE inhibitor, angiotensin-2 receptor blocker, beta blocker, mineralocorticoid receptor antagonist, diuretic, ivabradine, sacubit Lil valsartan, hydralazine with nitrate, and digoxin, the method. A method for identifying a subject at increased risk of developing a metabolic disorder, the method comprising:
determining or determining the presence or absence of an INHBE variant nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide in a biological sample obtained from the subject;
If the subject has INHBE criteria, the subject has an increased risk of developing a metabolic disorder; and
wherein if the subject is heterozygous or homozygous for the INHBE variant nucleic acid molecule, the subject has a reduced risk of developing a metabolic disorder. A method for identifying a subject having an increased risk of developing cardiovascular disease, the method comprising:
determining or determining the presence or absence of an INHBE variant nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide in a biological sample obtained from the subject;
If the subject has INHBE criteria, the subject has an increased risk of developing cardiovascular disease; and
wherein if the subject is heterozygous or homozygous for the INHBE variant nucleic acid molecule, the subject has a reduced risk of developing cardiovascular disease. 81. The method of claim 79 or 80, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule. 82. The method of claim 81, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule in the biological sample. 81. The method of claim 79 or 80, wherein the INHBE variant nucleic acid molecule is an mRNA molecule. 84. The method of claim 83, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample. 81. The method of claim 79 or 80, wherein the INHBE variant nucleic acid molecule is a cDNA molecule generated from an mRNA molecule. 86. The method of claim 85, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of an INHBE cDNA molecule generated from mRNA molecules in the biological sample. 81. The method of claim 79 or 80, wherein the INHBE variant nucleic acid molecule is a missense variant, a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant or an in-frame indel variant. , or a variant encoding a truncated INHBE polypeptide. 81. The method of claim 79 or 80, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide. 89. The method of any one of claims 79-88, wherein the determining step comprises sequencing total nucleic acid molecules in the biological sample. 90. The method of any one of claims 79 to 89, wherein the determining step
a) amplifying at least a portion of a nucleic acid molecule encoding an INHBE polypeptide;
b) labeling the amplified nucleic acid molecule with a detectable label;
c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and
d) detecting the detectable label. 91. The method of claim 90, wherein the nucleic acid molecule in the sample is an mRNA and the mRNA is reverse transcribed into cDNA prior to the amplification step. 90. The method of any one of claims 79 to 89, wherein the determining step
contacting a nucleic acid molecule in a biological sample with an alteration-specific probe comprising a detectable label; and
detecting the detectable label. 93. The method of any one of claims 79-92, wherein the determining step is performed in vitro. 94. The method of any one of claims 79-93, wherein the subject is on an INHBE basis and the subject is administered a standard dose of a therapeutic agent that treats or inhibits a metabolic disorder or cardiovascular disease and is administered an INHBE inhibitor. . 94. The therapeutic agent of any one of claims 79-93, wherein the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide, and the subject treats or inhibits a metabolic disorder or cardiovascular disease. is administered in an amount equal to or less than the standard dose, and the INHBE inhibitor is administered. 96. The method of claim 94 or 95, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. 96. The method of claim 94 or 95, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule. 98. The method of claim 97, wherein the Cas protein is Cas9 or Cpf1. 99. The method of claim 97 or 98, wherein the gRNA recognition sequence is located within SEQ ID NO: 1. 99. The method of claim 97 or 98, wherein the protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence. 101. The method of any one of claims 97-100, wherein the gRNA comprises from about 17 to about 23 nucleotides. 101. The method of any one of claims 97-100, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27. 96. The method of claim 94 or 95, wherein the metabolic disorder is type 2 diabetes, and the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone , pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, semaglutide, canagliflozin, dapagliflozin and empagliflozin or any combination thereof Method selected from. 96. The method of claim 94 or 95, wherein the metabolic disorder is obesity and the treatment is selected from orlistat, phentermine, topiramate, bupropion, naltrexone and liraglutide or any combination thereof. 96. The method of claim 94 or 95, wherein the metabolic disorder is elevated triglycerides and the therapeutic agent is rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibric acid, niacin and omega-3 fatty acids or any of these A method selected from a combination. 96. The method of claim 94 or 95, wherein the metabolic disorder is lipodystrophy, and the therapeutic agent is tesamorelin, metformin, poly-L-lactic acid, calcium hydroxyapatite, polymethylmethacrylate, bovine collagen, human collagen, silicone , and hyaluronic acid or any combination thereof. 96. The method according to claim 94 or 95, wherein the metabolic disorder is liver inflammation, and the therapeutic agent is a hepatitis treatment agent or a hepatitis vaccine. 96. The method of claim 94 or 95, wherein the metabolic disorder is fatty liver disease and the subject undergoes bariatric surgery and/or dietary intervention. 96. The method of claim 94 or 95, wherein the metabolic disorder is hypercholesterolemia, and the therapeutic agent is atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, colesevelam and selected from colestipol, alirocumab, evolocumab, niaspan, niacor, fenofibrate, gemfibrozil, and bempedoic or any combination thereof. 96. The method of claim 94 or 95, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statins and fiber, or any combination thereof. 96. The method of claim 94 or 95, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH), and the therapeutic agent is obeticholic acid, seloncertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E, arachidyl ami docolanoic acid, GS0976, emricasan, bolixivat, NGM282, GS9674, tropexor, MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, saroglitazar, BMS986036, ranifibranor, semaglutide, nita GRI_0621, EYP001, VK2809, Daymepen, LIK066, MT_3995, Elovisivats, Namodenosone, Poralumab, SAR425899, Sotagliflozin 6865571 NV 556, AZD2693, SP_1373, VK0214, HepaStem, TGFTX4, RLBN1127, GKT_137831, RYI_018, CB4209-CB4211, and JH_0920. 96. The method of claim 94 or 95, wherein the therapeutic agent for treating or inhibiting a metabolic disorder is a melanocortin 4 receptor (MC4R) agonist. 113. The method of claim 112, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule or small molecule. 114. The method of claim 113, wherein the protein is a peptide analog of MC4R. 114. The method of claim 113, wherein the peptide is setmelanotide. 113. The method of claim 112, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. 114. The method of claim 113, wherein the small molecule is 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid. 113. The method of claim 112, wherein the MC4R agonist is ALB-127158(a). 96. The method of claim 94 or 95, wherein the cardiovascular disease is hypertension, and the therapeutic agent is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol , bisoprolol fumarate, carteolol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, perrin Dopril, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, bepridil, diltiazem hydrochloride, felodipine, isradipine, nicardipine, nifedipine, nisoldipine, verapamil hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol labetalol hydrochloride, alpha methyldopa, clonidine hydrochloride, old anabenz acetate, guanfacine hydrochloride, guanadrel, guanethidine monosulfate, reserpine, hydralazine hydrochloride and minoxidil or any combination thereof. 96. The method of claim 94 or 95, wherein the cardiovascular disease is cardiomyopathy, and the therapeutic agent is an ACE inhibitor, angiotensin II receptor blocker, beta blocker, calcium channel blocker, digoxin, antiarrhythmic agent, aldosterone blocker, diuretic, anticoagulant, blood thinner and a corticosteroid. 96. The method of claim 94 or 95, wherein the cardiovascular disease is heart failure, and the therapeutic agent is an ACE inhibitor, angiotensin-2 receptor blocker, beta blocker, mineralocorticoid receptor antagonist, diuretic, ivabradine, sacubitril valsartan, nitrate containing hydralazine, and digoxin, a method. A therapeutic agent that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having:
an INHBE variant genomic nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide;
INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or
INHBE variant cDNA molecules encoding INHBE predicted loss-of-function polypeptides. 123. The method of claim 122, wherein the metabolic disorder is type 2 diabetes, and the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, theta selected from gliptin, saxagliptin, linagliptin, exenatide, liraglutide, semaglutide, canagliflozin, dapagliflozin and empagliflozin or any combination thereof; remedy. 123. The therapeutic agent of claim 122, wherein the metabolic disorder is obesity and the therapeutic agent is selected from orlistat, phentermine, topiramate, bupropion, naltrexone and liraglutide or any combination thereof. 123. The method of claim 122, wherein the metabolic disorder is elevated triglycerides and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibric acid, niacin and omega-3 fatty acids or any combination thereof. , the cure. 123. The method of claim 122, wherein the metabolic disorder is lipodystrophy and the therapeutic agent is tesamorelin, metformin, poly-L-lactic acid, calcium hydroxyapatite, polymethylmethacrylate, bovine collagen, human collagen, silicone, and hyaluronic acid. or any combination thereof. 123. The method of claim 122, wherein the metabolic disorder is liver inflammation and the treatment is a hepatitis treatment or hepatitis vaccine. 123. The method of claim 122, wherein the metabolic disorder is fatty liver disease and the subject undergoes bariatric surgery and/or dietary intervention. 123. The method of claim 122, wherein the metabolic disorder is hypercholesterolemia, and the therapeutic agent is atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, colesevelam and colestipol, selected from alirocumab, evolocumab, niaspan, niacor, fenofibrate, gemfibrozil, and bempedoic or any combination thereof. 123. The method of claim 122, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statins and fiber, or any combination thereof. 123. The method of claim 122, wherein the metabolic disorder is non-alcoholic steatohepatitis (NASH), and the treatment is obeticholic acid, seloncertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E, arachidylamidocolanoic acid, GRI_06 21 , EYP001, VK2809, nalmefene, LIK066, MT_3995, elobisibat, namodenoson, poralumab, SAR425899, sotagliflozin, EDP_305, isosabutate, gemcabene, TERN_101, KBP_042, PF_06865571, DUR928, PF_0 6835919 , NGM313, BMS_986171, Namacizumab, CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, Seladepar, PXL770, TERN_201, NV556, AZD2693, SP_1373, VK0214, Hephastem, TGFTX4, RLBN1127, GKT_137831, RYI_018, CB4209-CB4211, and JH_0920. 123. The therapeutic agent of claim 122, wherein the therapeutic agent that treats or inhibits the metabolic disorder is a melanocortin 4 receptor (MC4R) agonist. 133. The therapeutic agent of claim 132, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule or small molecule. 134. The therapeutic agent of claim 133, wherein the protein is a peptide analog of MC4R. 134. The therapeutic agent of claim 133, wherein the peptide is setmelanotide. 133. The therapeutic agent of claim 132, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. 134. The therapeutic agent of claim 133, wherein the small molecule is 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid. 133. The therapeutic of claim 132, wherein the MC4R agonist is ALB-127158(a). A therapeutic agent that treats or inhibits cardiovascular disease for use in the treatment of cardiovascular disease in a subject having:
an INHBE variant genomic nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide;
INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or
INHBE variant cDNA molecules encoding INHBE predicted loss-of-function polypeptides. 140. The method of claim 139, wherein the cardiovascular disease is hypertension and the treatment is chlorthalidone, chlorothiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol, bisoprolol Fumarate, carteolol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quina Pril hydrochloride, ramipril, trandolapril, candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, bepridil, diltiazem hydrochloride, felodipine , isradipine, nicardipine, nifedipine, nisoldipine, verapamil hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol labetalol hydrochloride, alpha methyldopa, clonidine hydrochloride, guanabenz acetate, A therapeutic agent selected from guanfacine hydrochloride, guanadrel, guanethidine monosulfate and reserpine, hydralazine hydrochloride and minoxidil or any combination thereof. 140. The method of claim 139, wherein the cardiovascular disease is cardiomyopathy and the treatment is an ACE inhibitor, angiotensin II receptor blocker, beta blocker, calcium channel blocker, digoxin, antiarrhythmic agent, aldosterone blocker, diuretic, anticoagulant, blood thinner and corticosteroid. , the cure. 140. The method of claim 139, wherein the cardiovascular disease is heart failure and the treatment is selected from the group consisting of ACE inhibitors, angiotensin-2 receptor blockers, beta blockers, mineralocorticoid receptor antagonists, diuretics, ivabradine, sacubitril valsartan, hydralazine with nitrates. , and digoxin, a therapeutic agent. An inhibin subunit beta E (INHBE) inhibitor that treats or inhibits a metabolic disorder for use in the treatment of a metabolic disorder in a subject having:
an INHBE variant genomic nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide;
INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or
INHBE variant cDNA molecules encoding INHBE predicted loss-of-function polypeptides. An inhibin subunit beta E (INHBE) inhibitor for use in the treatment and/or prevention of a metabolic disorder in a subject that is:
a) is referenced to an inhibin subunit beta E (INHBE) genomic nucleic acid molecule, an INHBE mRNA molecule, or an INHBE cDNA molecule; or
b)
i) an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide;
ii) an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or
iii) is heterozygous for an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide. 145. The INHBE inhibitor of claim 144, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. 145. The INHBE inhibitor of claim 144, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence in an INHBE genomic nucleic acid molecule. 147. The INHBE inhibitor according to claim 146, wherein the Cas protein is Cas9 or Cpf1. 148. The INHBE inhibitor of claim 146 or 147, wherein the gRNA recognition sequence is located within SEQ ID NO: 1. 148. The INHBE inhibitor of claim 146 or 147, wherein the protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence. 150. The INHBE inhibitor of any one of claims 146-149, wherein the gRNA comprises from about 17 to about 23 nucleotides. 150. The INHBE inhibitor according to any one of claims 146 to 149, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27. An inhibin subunit beta E (INHBE) inhibitor that treats or inhibits cardiovascular disease for use in the treatment of cardiovascular disease in a subject having:
an INHBE variant genomic nucleic acid molecule encoding an inhibin subunit beta E (INHBE) predicted loss-of-function polypeptide;
INHBE variant mRNA molecules encoding INHBE predicted loss-of-function polypeptides; or
INHBE variant cDNA molecules encoding INHBE predicted loss-of-function polypeptides. An inhibin subunit beta E (INHBE) inhibitor for use in the treatment and/or prevention of cardiovascular disease in a subject that is:
a) is a reference to an inhibin subunit beta E (INHBE) genomic nucleic acid molecule, an INHBE mRNA molecule, or an INHBE cDNA molecule; or
b)
i) an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide;
ii) an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or
iii) is heterozygous for an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide. 154. The INHBE inhibitor of claim 153, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. 154. The INHBE inhibitor of claim 153, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence in an INHBE genomic nucleic acid molecule. 156. The INHBE inhibitor according to claim 155, wherein the Cas protein is Cas9 or Cpf1. 157. The INHBE inhibitor of claim 155 or 156, wherein the gRNA recognition sequence is located within SEQ ID NO: 1. 157. The INHBE inhibitor of claim 155 or 156, wherein the protospacer adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence. 159. The INHBE inhibitor of any one of claims 155-158, wherein the gRNA comprises from about 17 to about 23 nucleotides. 159. The INHBE inhibitor according to any one of claims 155 to 158, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs: 9-27.

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